CN108463553A - Recombinant serine protease - Google Patents

Abstract

The present invention relates to the recombinant proteins including serine protease polypeptide, the serine protease polypeptide has serine protease in the presence of serpin, and can for example reverse the effect of serpin completely or partially in the subject treated with serpin.More specifically, this document describes the recombinant protein of the blood coagulation resisting function for reversing Coagulative inhibitors agent completely or partially and methods.

Description

Recombinant serine protease

Technical field

The present invention is in formulation art for medical purpose.More particularly it relates to specific recombinant serine Protease has serine protease in the presence of serpin.

Background technology

It is currently being deployed the serpin for being continuously increased quantity, prevents or inhibits serine protease Carry out its proteinase activity.Serine protease (or serine endopeptidase) is the enzyme of the peptide bond in scinderin, wherein Serine serves as nucleophilic amino acid (Hedstrom, 2002.Chem Rev 102 at proteinase activity site：4501- 4524).In human body, serine protease is responsible for coordinating various physiology courses, including digestion, immune response, blood coagulation and reproduction (Hedstrom, 2002.Chem Rev 102：4501-4524).Some well-known serine proteases include fibrin ferment, Plasma thromboplastin antecedent a, urokinase-type plasminogen activator and trypsase, first two participate in blood coagulation.

Serine protease can be inhibited by different types of inhibitor, including synthesis chemical inhibitor and native protein inhibit Agent.The referred to as family day of " serpins " (abbreviation of serpin (serine protease inhibitor)) Right inhibitor can form covalent bond with serine protease, to inhibit its function.The serine protease of most study inhibits Some in agent are antithrombase and alpha1-antitrypsin, by its respectively the effect in blood coagulation and pulmonary emphysema and be people institute Know.

The direct serpin of such as direct thrombin inhibitor (DTI) is being developed, and due to it Curative effect is rapid, is easy to be administered and need not monitor, it is therefore contemplated that it can replace such as anticoagulation on a large scale in the near future Classical oral anticoagulant (He et al., the 2015.Molecules 20,11046-11062 of enzyme；Wang et al., 2015.Arch Pharm 348：595-605).These inhibitor target the active site of serine protease, and be generally suitable for taking orally to The small molecule of medicine.Monovalent DTI further include inter alia argatroban, melagatran or its prodrug, ximelagatran or its before Medicine, dabigatran or its prodrug, dabigatran etcxilate (Dabigatran Etexilate) or its analog, peptide or peptidomimetic (peptidomimetic) inhibitor (Mehta et al., 2014.Expert Opin Ther Pat 24：47-67)、RWJ- 671818 or its analog (Lu et al., 2010.J Med Chem 53：1843-1856), 3- (2- PhenethyIaminos) -6- first Base -1- (2- amino -6- methyl -5- methylene amide ylmethyls pyridyl group) pyrazinones or its analog, (Sanderson et al., 1998.J Med Chem 41：4466-4474), (E)-N- (3- ((1- (benzo [b] thiophene -2- ylmethyls) -1H-1,2,3- three Azoles -4- bases) methoxyl group) phenyl) -2- (3- chlorphenyls) ethenesulfonamides or its analog (Siles et al., 2011.Bioorg Med Chem Lett 21：5305-5309), N- { 3- [(3- luorobenzyls) oxygroup] phenyl } -1- pyridin-4-yl piperidines -4- formyls Amine or its analog (de Candia et al., 2013.J Med Chem 56：8696-8711) and the compound of Merck 2 or Its analog (Morrissette et al., 2004.Bioorg Med Chem Lett 14：4161-4164).Monovalent DTI is It is specifically designed as that its proteinase activity is combined closely and stopped with the active site of fibrin ferment.

The unit price directly inhibitor of plasma thromboplastin antecedent a has also been found, and further includes 4,5,6- tri- inter alia Substituted pyrimidine derivatives (United States Patent (USP) US8609676 B2), BMS-262084 (Schumacher et al., 2007.Eur J Pharmacol 570：167-174), the compound 1 of Bristol-Meyers Squibb or its analog (Quan et al., J Med Chem 2014.57：955-969), the compound 2 of Bristol-Meyers Squibb and 33 or its analog (Pinto Et al., 2015.Bioorg Med Chem Lett 25：1635-1642), the compound 13 or its analog of AstraZeneca (Fjellstrom et al., 2015.PLoS One 10：E0113705), aryl boric acid (Lazarova et al., 2006.Bioorg Med Chem Lett 16：5022-5027) and Macrocyclic indole (Hanessian et al., 2010.Bioorg Med Chem Lett 20：6925-6928).

The monovalent inhibitor of urokinase-type plasminogen activator (activation, activator) also wraps inter alia Include WX-UK1 or its prodrug Upamostat, also referred to Mesupron or WX-671 or its analog and APC-10302 or its Analog (Katz et al., 2001.Chem Biol 8：1107-1121).What is interesting is, it has been found that direct thrombin inhibitor Melagatran and direct urokinase-type plasminogen activator inhibitor APC-10302 also can be with the active sites of trypsase In conjunction with and inhibit its serine protease (Dullweber et al. 2001.J Mol Biol 313：593-614；Katz etc. People, 2001.Chem Biol 8：1107-1121.

The disadvantage is that, it is usual effectively to restore normal serine protease using one of serpin It needs to replace serine protease completely, or inhibitor is effectively removed from subject.This is a disadvantage, due in inhibition Inducing the activity of serine protease preferably should be in clinical setting and in non-clinical afterwards can be immediately and directly real Existing, rather than as the time is gradually realized.For example, in the case of anticoagulant therapy, generally lack specific reverse Strategy can lead to the hemorrhage complication of potential threat to life after application inhibitor (such as inhibitor of fibrin ferment).The latter By taking following facts as an example：Only in Holland, the patient for receiving anticoagulant treatment per year over 5,000 suffers from and serious has Harmful bleeding episode, including (the Adriaansen H. et al. of the death more than 800：“Samenvatting Medische Jaarverslagen van de Federatie van Nederlandse Trombosediensten ", 2014；1-38).

Currently, the Reversal Strategy for directly and immediately preventing and stopping the inhibiting effect of serpin is not looked for yet It arrives.

Invention content

The present invention by provide include selected from include fibrin ferment, plasma thromboplastin antecedent a, urokinase-type plasminogen activator and The recombinant protein of the serine protease polypeptide of the group of trypsase is as the suppression for preventing and stopping serpin The appropriate Reversal Strategy of making solves the problems, such as this, and the polypeptide includes at least one amino acid in outer polypeptide surface structure The insertion of residue, wherein the peptide structure is：SEQ ID NO:Between 1 Gly-427 and Asp-462, preferably His-450 with Amino acid residue region between Asp-462, between more preferable His-450 and Leu-459；SEQ ID NO:2 Val-463 with Amino acid residue region between Asp-480, between preferably His-469 and Asp-480 or Ser-477；SEQ ID NO:3 Between Leu-73 and Asp-107, between preferably His-76 and Asp-107, between more preferable Gln-87 and Asp-107, most preferably Amino acid residue region between His-96 and Leu-104 or Asp-107；SEQ ID NO:4 Val-237 and Asp-275 it Between, between preferably Phe-254 or Val-256 and Asp-275 or Asn-274, more preferable His-262 and Asp-275, Asn-274 Or the amino acid residue region between Ala-271.

Preferably, serine protease polypeptide is selected from fine by fibrin ferment, plasma thromboplastin antecedent a, trypsase and urokinase-type The group of plasminogen activator composition, is preferably selected from by human thrombin, human blood coagulation XIa, people's trypsase and human urokinase class The group of activator of plasminogen composition.It should be noted that SEQ ID NO:1 shows the amino acid sequence of human thrombin original, SEQ ID NO:2 show the amino acid sequence of human blood coagulation XI, SEQ ID NO:3 show the amino acid sequence of people's trypsase -1, SEQ ID NO:4 show the amino acid sequence of human urokinase type plasminogen activator.

It has been found that with the amino acid composition changed, preferably in the SEQ ID NO of fibrin ferment:1 Gly-427 with In region between Asp-462, in the SEQ ID NO of plasma thromboplastin antecedent a:Between 2 Val-463 and Asp-480 or Ser-477 Region in, in the SEQ ID NO of trypsase:In region between 3 Leu-73 and Asp-107 or in urokinase-type fibrinolytic The SEQ ID NO of activation of zymogen object:Insertion at least one amino acid in region between 4 Val-237 and Asp-275 The serine egg selected from the group formed by fibrin ferment, plasma thromboplastin antecedent a, trypsase and urokinase-type plasminogen activator White enzyme polypeptide has catalytic activity in the presence of serpin.This is surprising, due to obtainable Show that the amino acid in the peptide structure of outer surface is residual with the crystal structure of the serine protease in the compound of its inhibitor composition Base is not in contact with inhibitor (referring to (for example) Fig. 2-Fig. 4).

In addition, the amino acid residue in the peptide structure of outer surface seems that flexible ring (loop) structure can be formed, its composition is (i.e. The identity (type, identity) of amino acid residue) and amino acid residue quantity in fibrin ferment, human blood coagulation XIa, people's pancreas egg White is not conservative between enzyme and human urokinase type plasminogen activator.By being inserted at least one amino acid residue to this The change of ring is considered will not changing serine stretch protein after the combination of inhibitor and serine protease and/or binding inhibitors The activity of enzyme.

In addition, the known suppression of fibrin ferment, human blood coagulation XIa, people's trypsase and urokinase-type plasminogen activator Preparation is the incoherent compound in structure.This by experience further such that can not possibly be speculated in fibrin ferment, coagulation factor It is inserted into the outer surface peptide structure of Xia, trypsase or urokinase-type plasminogen activator serine protease at least one Amino acid residue generates the inhibition to inhibitor with the serine protease of the sensibility reduced.

Compared with the serine protease formed without the amino acid of the change, recombinant serine protease is to silk ammonia The inhibition of pepsin inhibitor is with the sensibility reduced.Therefore, the present invention provides the removing toxic substances of serpin Agent independent of the generation of free endogenous serine protease, and provides quickly and directly prevents and stop silk ammonia The Reversal Strategy of the inhibiting effect of pepsin inhibitor.

As used herein, term " serine protease " refer to by the enzyme of hydrolysising peptide key protein degradation, and its lead It is characterized in that in the active serine residue of active site.Serine protease is usually also referred to serine endopeptidase Enzyme.Preferably, term " serine protease " refers to catalytic triad amino acids residue histidine, asparatate and silk (it is by way of example in SEQ ID NO for propylhomoserin:His-406, Asp-462 and Ser-568 are expressed as in 1) spatial distribution Enzyme.Technical staff can easily assess and find the catalytic triad amino acids residue in serine endopeptidase.The art Language is included in the serine endopeptidase in (EC) 3.4.21 fermentoids, such as chymotrypsin, trypsase, blood coagulation Enzyme, proconvertin a, factor IXa, plasma thromboplastin antecedent a, elastoser, granzyme A, granzyme B, kallikrein 8, with And the precursor of these serine proteases, such as inactive preceding original-precursor (prepro-precursors) and before-precursor (pro-precursors).Serine protease polypeptide is preferably fibrin ferment, plasma thromboplastin antecedent a, trypsase or urokinase-type Activator of plasminogen polypeptide is preferably selected from and is swashed by fibrin ferment, plasma thromboplastin antecedent a, trypsase or urokinase-type plasminogen The group of being polypeptide composition.Determine whether a kind of albumen is that the method for serine protease is well known in the art, and Albumen enzyme detection kit including alignment and use such as Sigma-Aldrich.

The amino acid sequence of human thrombin original is in SEQ ID NO:It is shown in 1, and can beIn It is found under accession number (registration number, accession number) AAC63054.1.With SEQ ID NO:Listed sequence is solidifying in 1 Hemase original is containing preceding original-targeting sequencing (SEQ ID NO:1 amino acid residue 1 to 43), subsequent correspond to activated peptide piece Sequence, the subsequent blood coagulation of section 1 (amino acid residue 44-198) and subsequent activation peptide fragment 2 (amino acid residue 199-327) The precursor of enzyme light chain (amino acid residue 328-363) and fibrin ferment heavy chain (amino acid residue 364-622).As used herein, Term " factor " refers to inactive PIVKA II.As used herein, term " fibrin ferment " refers to having The catalytic activity form of the factor of fibrin ferment light chain and heavy chain.According to definition used herein, factor includes solidifying Hemase polypeptide.

In the case of the present invention, if a kind of albumen is procoagulant serine protease, preferably cleavable fiber Arg- in proteinogen |-Gly keys are to form fibrin and discharge fibrinopeptide A and the procoagulant serine stretch protein of B Enzyme, then it is exactly factor or blood coagulation enzyme polypeptide.Fibrin ferment is also referred to fibrinogenase, preferably includes to correspond to difference The amino acid residue for the amino acid residue segment guarded between the factor or blood coagulation enzyme polypeptide of species (type, species) Segment.E.g., including the procoagulant serine protease of following polypeptide is considered as factor or blood coagulation enzyme polypeptide, described Polypeptide contains corresponding to SEQ ID NO:1 amino acid residue Arg-338 to Glu-343, Pro-376 to Leu-381, Ser- 385 to Leu-395, Arg-461 to Leu-465, Lys-498 to Leu-507, Lys-559 to Lys-575 and Gly-586 are arrived The amino acid residue segment of Arg-596.Term " factor " or " fibrin ferment " include the serine referred in EC 3.4.21.5 Protease.

Technical staff can for example by be suitble to the purpose substrate such as below carry out test proteins hydrolysis cutting come Determine whether a kind of serine protease is actually fibrin ferment：(i) S-2238 of Chromogenix^TMSubstrate (brand of Instrumentation Laboratory (Bedford, MA, USA), chemical formula are Bz-IIe-Glu (γ-OR)- Gly-Arg-pNAHCl (R=H (50%) and R=CH3 (50%)；Molecular weight：741.3；Item number：82 0,316 39), are used for Fibrin ferment, while according to the explanation of manufacturer；And/or (ii)TH series of chromogenic substrates (DSM Nutritional Products, CH), chemical formula is：Tos-Gly-Pro-Arg-pNA·AcOH、H-D-CHG-Ala-Arg- pNA·2AcOH、H-D-CHG-But-Arg-pNA·2AcOH、H-D-CHG-Pro-Arg-pNA·2AcOH、H-D-CHA-Ala- Arg-pNA2AcOH、H-D-CHA-Gly-Arg-pNA·2AcOH、CH₃OCO-Gly-Pro-Arg-pNAAcOH and/or H- beta-Ala-Gly-Arg-pNA·2AcOH。

The amino acid sequence of human blood coagulation XI is in SEQ ID NO:It is shown in 2, and can beIn It is found at accession number AAA51985.1.With SEQ ID NO:The plasma thromboplastin antecedent of sequence shown in 2 is containing signal peptide (amino acid is residual for (amino acid residue 1-18), plasma thromboplastin antecedent a heavy chains (amino acid residue 19-387) and plasma thromboplastin antecedent a light chains Base 388-625) precursor protein.As used herein term " plasma thromboplastin antecedent " refers to inactive plasma thromboplastin antecedent precursor Albumen.As used herein, term " plasma thromboplastin antecedent a " refers to the catalytic activity form of plasma thromboplastin antecedent, with blood coagulation because Sub- XIa heavy chains and plasma thromboplastin antecedent a light chains.According to definition used herein, plasma thromboplastin antecedent includes plasma thromboplastin antecedent a more Peptide.

In the case of the present invention, if a kind of albumen is the Arg- in selectivity cutting factors IX |-Ala keys and Arg- |-Val keys are to form the procoagulant of factors IX a, then it is just considered as plasma thromboplastin antecedent or plasma thromboplastin antecedent a polypeptides.It is described The full length amino acid sequence of albumen preferably include corresponding to different plant species plasma thromboplastin antecedent polypeptide or plasma thromboplastin antecedent a polypeptides it Between guard amino acid residue segment amino acid residue segment.E.g., including containing corresponding to SEQ ID NO:2 amino acid The amino acid residue segment of residue A sp-480 to Ala-482, Cys-560 to Gly-562 and Gly-573 to Leu-579 The procoagulant serine protease of polypeptide is considered as plasma thromboplastin antecedent or plasma thromboplastin antecedent a polypeptides.Term " coagulation factor XI " or " plasma thromboplastin antecedent a " include the serine protease referred in EC 3.4.21.27.Technical staff can be for example by suitable The substrate such as below for closing the purpose carries out test proteins hydrolysis cutting to confirm that a kind of serine protease is coagulation factor XI or plasma thromboplastin antecedent a polypeptides：(i) S-2366 of Chromogenix^TMSubstrate (Instrumentation Laboratory The brand of (Bedford, MA, USA), chemical formula are pyroGlu-Pro-Arg-pNAHCl (molecular weight 539.0；Item number (Part Number) is 82 109039), while according to the explanation of manufacturer；And/or (ii)FXIa develops the color Substrate (DSM Nutritional Products, CH), chemical formula are Z-Aad-Pro-Arg-pNA AcOH).

The amino acid sequence of people's trypsase -1 is in SEQ ID NO:It is shown in 3, and can beIn It is found at " NP_002760.1 ".With SEQ ID NO:The trypsase -1 of sequence shown in 3 is with signal peptide (ammonia Base acid residue 1-15), activated peptide (amino acid residue 16-23), α-trypsase chain 1 (amino acid residue 24-122) and α-pancreas egg The precursor protein of white enzyme chain 2 (amino acid residue 123-247).Double-stranded form passes through in SEQ ID NO:3 residue A rg-122 it Proteolysis cutting is carried out afterwards to generate.It should be noted that α-trypsase chain can be used as a kind of peptide chain (amino acid residue of catalytic activity 24-247) exist.It is generally known that trypsase is the prototype of serine protease.As used herein, term " pancreas egg White enzyme " refers to inactive trypsase precursor protein, and the single stranded form of catalytic activity and the double-stranded form of catalytic activity have The α of separation-trypsase chain 1 and α-trypsase chain 2.

In the case of the present invention, if a kind of albumen is preferred cutting Arg- |-Xaa, Lys- | the serine egg of-Xaa White enzyme, then it be just considered as trypsase polypeptide.The full length amino acid sequence of the albumen preferably includes to correspond to different plant species Trypsase polypeptide between guard amino acid residue segment amino acid residue segment.Such as correspond to SEQ including containing ID NO:3 amino acid residue Phe-47 to Gly-50, Gly-191 to Gln-197, Asp-199 to Pro-203 and Val- The serine protease of the polypeptide of 214 to Gly-217 amino acid residue segment is considered as trypsase polypeptide.Term " pancreas Protease " includes preferred cutting Arg- |-Xaa, Lys- |-Xaa's and/or EC 3.4.21.4 in the serine protease listed. Therefore term " trypsase " includes the trypsase albumen referred to except trypsase 1, such as trypsase 2, tryptose Enzyme 3, trypsase 3, trypsase 4, trypsase 5 and trypsase 6.The trypsase is preferably trypsase 1.Technology Personnel can be for example by confirming a kind of ammonia to being suitble to the substrate such as below of the purpose to carry out test proteins hydrolysis cutting Whether pepsin is trypsase：(i) S-2222 of Chromogenix^TMSubstrate (Instrumentation Laboratory The brand of (Bedford, MA, USA)), chemical formula is Bz-Ile-Glu (γ-OR)-Gly-Arg-pNAHCl (R=H (50%) And R=CH₃(50%), molecular weight 741.3, catalog number (Cat.No.) S820316, while according to the explanation of manufacturer；And/orTRY (trypsase) chromogenic substrate (DSM Nutritional Products, CH), chemical formula Cbo- Val-Gly-Arg-pNA·AcOH。

The amino acid sequence of human urokinase type plasminogen activator is in SEQ ID NO:It is shown in 4, and can beIn found at " AAK53822.1 ".With SEQ ID NO:The urokinase-type fibrinolytic of sequence shown in 4 Activation of zymogen object is the precursor protein for having signal peptide (amino acid residue 1-20) and chain (amino acid residue 21-431), the chain (amino acid residue 21-431) can be further divided into long-chain A (amino acid residue 21-177), short chain A (amino acid residue 156-177) and Chain B (amino acid residue 179-431).As used herein, term " urokinase-type plasminogen activator " refers to inactive Urokinase-type plasminogen activator precursor protein and its catalytic activity chain form.

In the case of the present invention, if a kind of albumen is the Arg- in specificity cutting plasminogen |-Val keys are with shape At the serine protease of fibrinolysin, then it is just considered as urokinase-type plasminogen activator.The overall length amino of the albumen Acid sequence preferably includes the amino acid residue guarded between the urokinase-type plasminogen activator polypeptide corresponding to different plant species The amino acid residue segment of segment.E.g., including containing corresponding to SEQ ID NO:4 amino acid residue His-119 to Asn- The amino acid residue segment and the Arg- in specificity cutting plasminogen of 124 and/or Asn-274 to Leu-278 |-Val keys Serine protease to form the polypeptide of fibrinolysin is considered as urokinase-type plasminogen activator polypeptide.

Technical staff can for example by be suitble to the purpose substrate such as below carry out test proteins hydrolysis cutting come Confirm whether serine protease is urokinase-type plasminogen activator：(i) S-2444 of Chromogenix^TMSubstrate (brand of Instrumentation Laboratory (Bedford, MA, USA)), chemical formula Glu-Gly-Arg-pNAHCl (molecular weight 498.9), while according to the explanation of manufacturer；And/or (ii) is used for urokinase-type plasminogen activatorUPA- series of chromogenic substrate (DSM Nutritional Products, CH), chemical formula Bz-beta- Ala-Gly-Arg-pNAAcOH and/or Cbo-Glu (OtBu)-Gly-Arg-pNA AcOH.

As used herein, term " recombinant (recon, recombinant) " refers to having used those skilled in the art The albumen that the recombinant DNA technology known generates.Recombinant protein is preferably since such as amino acid composition difference is (due to amino acid residue Exchange and/or the missing of one or more amino acid residue or insertion and cause) and/or due to being repaiied after such as glycosylated translation The difference of decorations and it is different from native protein.

As used herein, phrase " recombinant protein for including serine protease " is intended to cover include recombinant serine egg White enzyme polypeptide, the recombinant serine protease polypeptide for being preferably derived from mammal, the weight for more preferably deriving from primate Group serine protease polypeptide and most preferably derive from people recombinant serine protease polypeptide albumen.The phrase includes Such as the mammal serine protease precursor protein of recombination, such as factor, it is processed and/or activates and moved for lactation Object blood coagulation enzyme polypeptide.Therefore, albumen of the invention be preferably recombinate mammal, preferably primate, more preferably People's or humanization fibrin ferment, plasma thromboplastin antecedent a, trypsase or urokinase-type plasminogen activator, in outer surface Peptide structure includes the insertion of at least one amino acid residue, wherein the peptide structure is：Corresponding to the SEQ ID of fibrin ferment NO:Between 1 Gly-427 and Asp-462, between preferably His-450 and Asp-462, more preferable His-450 and Leu-459 it Between amino acid residue region amino acid residue region；Corresponding to the SEQ ID NO of plasma thromboplastin antecedent a:2 Val-463 with The amino acid residue area in the amino acid residue region between Asp-480, between preferably His-469 and Asp-480 or Ser-477 Domain；Corresponding to the SEQ ID NO of trypsase:Between 3 Leu-73 and Asp-107, preferably His-96 and Asp-107 or Leu- The amino acid residue region in the amino acid residue region between 104；And the SEQ corresponding to urokinase-type plasminogen activator ID NO:Between 4 Val-237 and Asp-275, between preferably Phe-254 or Val-256 and Asp-275 or Asn-274, it is more excellent Select the amino acid residue region in the region between His-262 and Asp-275 or Asn-274.In addition, the phrase includes such Albumen：It further includes one or more other amino acid sequences other than serine protease polypeptide, such as is constituted a kind of The amino acid sequence and/or one or more other identification peptides of label (such as FLAG labels as described in EP0150126).

As used herein, term " humanization " refers to replacing the external amino acid residue of the albumen of preferably one species Change or humanization be the albumen people's homologue present in amino acid residue, so that the albumen of the first species is applied to people When not will produce immunogenicity (immunogenic), or generate less immunogenicity.External residues are replaced preferably to inside Contacting hardly to have structural domain or domain between light chain and heavy chain influences, or without influencing.From non- People is preferably derived from mammal, is more preferably preferably humanized from the albumen of the present invention of primate, so as to when giving People reduces the immunogenicity of the albumen when applying.

The non-human protein of the present invention preferably includes the mammal serine protease polypeptide of humanization, more preferable humanization Primate serine protease polypeptide, this is because the risk of expected antigen response when applying in human body with include non- The albumen of the present invention of the serine protease polypeptide of humanization is compared to lower.

In the case of humanized proteins, it can be appreciated that can be applied to the humanization approach of antibody.This method utilizes existing By Kabat et al. (1987) Sequences of Proteins of Immunological Interest, 4^thEd., The human antibody of Bethesda, Md., National Institutes of Health (National Institutes of Health) editor can The sequence data in structure changes domain, to the database of the update of the database and other addressable the United States and abroads (nucleic acid and Albumen).Non-limiting examples for generating the method for humanized antibody include：EP 519596；U.S. Patent number 6,797, 492；And Padlan et al., 1991.Mol Immunol 28：489-498.The humanization approach of non-human protein is further lifted Example explanation, Sarkar et al., 2012, Journal of Lipids, article ID 610937, the 1-13 pages describes and passes through change The surface of enzyme is successfully by -1 humanization of paraoxonase, to reflect human sequence.

As used herein, term " serpin " is the medicine for referring to inhibit serine protease Agent.Preferably, serpin is the direct serpin of unit price, it is therefore preferable to small molecule or peptide Or peptidomimetic, it is combined and is worked by the active site with serine protease.Preferably, serpin is solidifying Thrombin inhibitor, plasma thromboplastin antecedent a inhibitor, trypsin inhibitor or urokinase-type plasminogen activator inhibitor.

As used herein, term " thrombin inhibitor " includes but not limited to direct thrombin inhibitor comprising all Such as the divalent direct thrombin inhibitor of hirudin, bivalirudin and lepirudin 023 ludon, pass through the active site with fibrin ferment In conjunction with working with being combined by the external site 1 with fibrin ferment.External site 1 cannot functionally connect in factor Closely, but (Huntington, 2005.J Thromb Haemos 3 will be exposed after the activation：1861-1872；Lane et al., 2005.Blood：106：2605-2612).Term " thrombin inhibitor " further comprises the unit price referred to directly fibrin ferment suppression Preparation, and preferably monovalent direct thrombin inhibitor, are combined by the active site with fibrin ferment and are worked.These lists Valence direct thrombin inhibitor includes：Argatroban ((2R, 4R) -1- [(2S) -5- (diamino methvleneamino) -2- [(3- first Base -1,2,3,4- tetrahydroquinoline -8- bases) sulfonamido] valeryl] -4- methyl-pi -2- formic acid) or its bioactivity it is similar Object；Melagatran (2- [[(1R) -2- [(2S) -2- [(4- carbamimido-phenyls) methylcarbamoyl] azetidine -1- bases] - 1- cyclohexyl -2- oxygen ethyl] amino] acetic acid) and its prodrug ximelagatran (2- [[(1R) -1- cyclohexyl -2- [(2S) -2- [[4- [(Z)-N'- hydroxyls amidino groups] phenyl] methylcarbamoyl] azetidine -1- bases] -2- oxygen ethyl] amino] ethyl acetate) Or its bioactive analogue；Dabigatran (3- [[2- [(4- amidino groups anilino-) methyl] -1- tolimidazole -5- carbonyls Base]-pyridine -2- bases amino] propionic acid) or its bioactive analogue, such as dabigatran etcxilate (dabigatran Etexilate) (3- [[2- [[4- [(Z)-N'- hexyloxy carbonyls amidino groups] anilino-] methyl] -1- tolimidazole -5- carbonyls Base]-pyridine -2- bases amino] ethyl propionate)；RWJ-671818 (1- { N- [2- (amidino groups amino oxygroup) ethyl] amino } carbonyl first Base -6- methyl -3- [bis- fluoro- 2- PhenethyIaminos of 2,2-] pyrazinones) or its bioactive analogue；3- (2- phenethyl ammonia Base) -6- methyl-1s-(2- amino -6- methyl -5- methylene amide ylmethyls pyridyl group) pyrazinones or its bioactivity it is similar Object；(E)-N- (3- ((1- (benzo [b] thiophene -2- ylmethyls) -1H-1,2,3- triazole-4-yls) methoxyl group) phenyl) -2- (3- Chlorphenyl) ethenesulfonamide or its bioactive analogue；And ((S)-N- (2- (amino methyl) -5- of compound 2 of Merck Chlorobenzyl) -1- ((R) -2- hydroxyl -3,3- dimethylbutanoyls) pyrrolidines -2- formamides) or its bioactive analogue.It is excellent Selection of land, direct thrombin inhibitor are monovalent direct thrombin inhibitor, are preferably suitable for the small molecule of oral medication, for example, Ah Add bent class, melagatran and its prodrug, ximelagatran or dabigatran and its prodrug dabigatran etcxilate (dabigatran ) or the bioactive analogue of these molecules etexilate.

Alternatively, direct thrombin inhibitor is peptide or peptidomimetic inhibitor (Mehta et al., 2014.Expert Opin Ther Pat 24：47-67).

As used herein, term " plasma thromboplastin antecedent a inhibitor " is the active site knot referred to plasma thromboplastin antecedent a Merge the inhibitor for inhibiting its proteinase activity.The term includes direct plasma thromboplastin antecedent a inhibitor, preferably and coagulation factor The active site of XIa combines and inhibits the small molecule of its proteinase activity.The group of direct plasma thromboplastin antecedent a inhibitor includes：4, 5,6- trisubstituted pyrimidine derivatives；BMS-262084 ((2S, 3R) -1- [4- (t-Butylcarbamoyl) piperazine -1- carbonyls Base] -3- [3- (diamino methvleneamino) propyl] -4- oxygroup azetidine -2- formic acid) or its bioactive analogue； (3'- [(2S, the 4R) -6- amidino groups -4- methyl 4-phenyl -1,2,3,4- tetrahydrochysenes of compound 1 of Bristol-Meyers Squibb Quinoline -2- bases] -4- carbamoyls -5'- [(3- methylbutyryls) amino] diphenyl -2- formic acid), 2 (trans--N- ((S) - 1- (4- (3- amino-1 h-indazole -6- bases) the chloro- 1H- imidazoles -2- bases of -5-) -2- phenethyls) -4- (amino methyl) hexamethylene first Amide) and 33 ((2E)-N- { (1S) -1- [4- (3- amino-1 h-indazole -6- bases) -1H- imidazoles -2- bases] -2- phenethyls } -3- [the chloro- 2- of 5- (1H-TETRAZOLE -1- bases) phenyl] propyl- 2- acrylamides) or its bioactive analogue；The chemical combination of AstraZeneca (N- [(1S) -1- benzyls -2- [(the chloro- 2- Oxy-1s H- quinolyl-4s of 6-) methylamino] -2- oxygroups-the ethyl] -4- hydroxyls of object 13 Base -2- Oxy-1 H- quinoline -6-carbo) or its bioactive analogue；Aryl boric acid；Macrocyclic indole；And peptide or peptidomimetic press down Preparation.Preferably, plasma thromboplastin antecedent a inhibitor is direct plasma thromboplastin antecedent a inhibitor, the preferably work with plasma thromboplastin antecedent a Property site combine and inhibit the small molecule of its proteinase activity, be preferably suitable for oral medication small molecule.

As used herein, term " urokinase-type plasminogen activator inhibitor " is to refer to and urokinase-type fibrinolytic The active site of activation of zymogen object combines and inhibits the inhibitor of its proteinase activity.The term includes direct urokinase-type fibrinolytic Activation of zymogen object inhibitor is preferably combined with the active site of urokinase-type plasminogen activator and inhibits its protease activity The small molecule of property.The group of direct urokinase-type plasminogen activator inhibitor further includes WX-UK1 (4- inter alia [(2S) -3- (3- carbamimido-phenyls) -2- [[2,4,6- tri- (propane -2- bases) phenyl] sulfonamido] propiono] piperazine -1- formic acid Ethyl ester) or its prodrug Upamostat, also referred to Mesupron or WX-671 (the latter：N α-(2,4,6- tri isopropyl benzenesulfonyls Base) -3- amidino groups-(L)-phenylalanine -4- ethoxycarbonylpiperazines), APC-10302 (6- chloro- 2- (2- Hydroxy-diphenyis - 3- yls) -1H- indoles -5- carbonamidines) or these compounds bioactive analogue.Alternatively, urokinase-type fibrinolysin Former activator inhibitor is peptide or peptidomimetic inhibitor.Preferably, direct urokinase-type plasminogen activator is that unit price is directly urinated Kinases plasminogen activator inhibitor is preferably suitable for the small molecule of oral medication, such as WX-UK1 and its prodrug Upamostat。

As used herein, term " trypsin inhibitor " is to refer to be combined and pressed down with the active site of trypsase Make the inhibitor of its proteinase activity.The term includes direct trypsin inhibitor, preferably with the active sites of trypsase Point combines and inhibits the small molecule of its proteinase activity.The group of direct trypsin inhibitor includes that melagatran and its prodrug are uncommon U.S. plus group (the latter：2- [[(1R) -1- cyclohexyl -2- [(2S) -2- [[4- [(Z)-N'- hydroxyls amidino groups] phenyl] methylamino first Acyl group] azetidine -1- bases] -2- oxygen ethyl] amino] ethyl acetate), APC-10302 (the chloro- 2- of 6- (2- hydroxyls-hexichol Base -3- bases) -1H- indoles -5- carbonamidines) and these molecules bioactive analogue.

As used herein, term " bioactive analogue " or " analog " refer to spreading out for pointed reference compound Biology or segment show biological function identical with reference compound and keep the target of such as anticoagulation or blood coagulation enhancing effect Activity.Therefore analog is preferably the analogue and functional analogue of pointed reference compound.

As used herein, term " homology " refers to the amino acid sequence identity between two kinds of amino acid sequences, table It is shown as the percentage of the same amino acid residue when comparing two kinds of amino acid sequences.The comparison is preferably in two kinds of amino acid sequences Total length on carry out.

As used herein, term " region " refers to using two conservative amino acid residues as the amino acid residue chain on boundary Section.The region includes two amino acid on the boundary for constituting the region.As the number of amino acid residue used herein is based on SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3 and SEQ ID NO:4 amino acid sequence.As used herein, art Language " amino acid region " include correspond to the delimited area (such as non-human thrombin, plasma thromboplastin antecedent a, trypsase and/or Region in urokinase-type plasminogen activator) amino acid residue region.It should be understood to the one skilled in the art that such as SEQ ID NO:1 In it is indicated, position of the region in for example non-human thrombin can be different from the position in human thrombin.But two For conservative amino acid residue in the both sides in indicated region, this can allow technical staff's identification to correspond to the institute in non-human protein State the region of delimited area.

As used herein, term " being inserted into (insertion) " or " (inserted) of insertion " refer in natural silk ammonia At least one amino acid residue is added in the specific region of pepsin polypeptide, to this with natural serine protease polypeptide Amino acid residue quantity in region is compared to the amino acid residue quantity increased in the region.

As used herein, term " replacing (replacement) " or " (replaced) of replacement " refer in serine Replace one or more amino acid residues in the specific region of protease polypeptide or at specific site, to change the region In amino acid sequence, but do not change amino acid residue quantity.Replacement is missing from an amino acid residue then in same position It is inserted into the result of a different aminoacids residue.

As used herein, term " missing (deletion) " or " (deleted) of missing " refer in serine stretch protein One or more amino acid residues are lacked in the specific region of enzyme polypeptide or at specific site, thus and natural serine protease Amino acid residue quantity in the region of polypeptide is compared to the amino acid residue quantity in the region for reducing the polypeptide.

As used herein, term " natural serine protease polypeptide " refers to that be naturally occurring in animal, preferably lactation dynamic Endogenous serine protease polypeptide in object, more preferable primate, more preferable people.

As used herein, term " amino acid composition " refers to the amino acid sequence and length of amino acid residue segment, Middle length is determined by the quantity of amino acid residue in the segment.

Be inserted into, replace and/or the one or more amino acid residues of missing, be preferably inserted into one or more amino acid residues can It is carried out using recombinant DNA technology well known to those skilled in the art.For example, synthetic DNA, PCR can be used in those skilled in the art Technology and molecular cloning, to obtain the recombinant dna construct with the DNA sequence dna for encoding albumen of the present invention.In Green and Sambrook, " Molecular Cloning:A Laboratory Manual ", CSHL Press, it is suitable to describe in 2012 Method and kit for.

As used herein, term " outer surface peptide structure " refers to continuous amino acid residue segment, also referred to peptide ring (peptide loop) or peptide bung flange (coil), appear in the natural fibrin ferment of folding, plasma thromboplastin antecedent a, trypsase and The outside of urokinase-type plasminogen activator polypeptide.Preferably, peptide structure is：Corresponding to the SEQ ID NO of fibrin ferment:1 Ammonia between Gly-427 and Asp-462, between preferably His-450 and Asp-462, between more preferable His-450 and Leu-459 The amino acid residue region of base acid full region；Corresponding to the SEQ ID NO of plasma thromboplastin antecedent a:2 Val-463 and Asp-480 Between, the amino acid residue region in amino acid residue region between preferably His-469 and Asp-480 or Ser-477；Correspond to The SEQ ID NO of trypsase:Between 3 Leu-73 and Asp-107, between preferably His-96 and Asp-107 or Leu-104 The amino acid residue region in amino acid residue region；And the SEQ ID NO corresponding to urokinase-type plasminogen activator:4 Val-237 and Asp-275 between, between preferably Phe-254 or Val-256 and Asp-275 or Asn-274, more preferable His- The amino acid residue region in the amino acid residue region between 262 and Asp-275 or Asn-274.

It has been found that institute in the outer surface peptide structure, preferably pointed by such as herein above and such as Fig. 2-Fig. 4 It is inserted at least one in the region of the fibrin ferment, plasma thromboplastin antecedent a, trypsase and the urokinase-type plasminogen activator polypeptide that show A amino acid residue will produce to serpin, preferably direct fibrin ferment, plasma thromboplastin antecedent a, trypsase or The albumen that the sensibility of the inhibition of urokinase-type plasminogen activator inhibitor reduces.

With for example corresponding to SEQ ID NO:Amino acid residue area between 1 His-450 and Asp-462 or Leu-459 Domain the relevant phrase in amino acid residue region " correspond to ... with ... between amino acid residue region " be used for herein It indicates to correspond to SEQ ID NO:The conservative His and Asp of 1 His-450 and Asp-462 or another fibrin ferment of Leu-459 The residue quantity of residue can with belong to SEQ ID NO:The residue quantity of His with the Asp residues in 1 is different.Amino acid The difference of residue quantity can be caused by the different numberings of such as amino acid residue.In addition, and in an illustrative manner, ammonia The different of base acid residue quantity can be by the length and SEQ ID NO of factor polypeptide:Human thrombin original polypeptide shown in 1 The difference of length cause.Similarly, when arranging a variety of factor polypeptides of multiple and different species, those skilled in the art It should readily recognize that, SEQ ID NO:1 amino acid residue Gly-427 is to protect between the factor polypeptide of different plant species It keeps.Therefore the amino acid residue of the amino acid residue in the serine protease corresponding to different plant species is identified It is possible.Therefore it will be understood by those skilled in the art that as numbering amino acid residues used herein not to the present invention into Row limitation, but just to for the sake of clear.

Technical staff should be appreciated that the SEQ ID how identified corresponding on the boundary for constituting a region as described herein NO:1、SEQ ID NO:2、SEQ ID NO:3 or SEQ ID NO:Amino acid residue between 4 conservative amino acid residues The amino acid residue region in region.Technical staff for example can directly confirm SEQ ID NO:1 His-450 and Asp-462 is it In the serine protease polypeptide of its species there is also highly conserved residue.

Due to SEQ ID NO:In 1 His-450 and Asp-462 or surrounding or non-human serine protease is more In peptide in corresponding His and Asp residues or surrounding amino acid residue region highly conserved property, people in the art Member can identify corresponding to SEQ ID NO:The amino acid residue in the amino acid residue region between 1 His-450 and Asp-462 Region.Same rule is suitable for constituting other amino acid residues on the boundary in a region as described herein.In other words, The conservative property of particular amino acid residue can be provided to technical staff about in inhuman serine protease polypeptide as described herein Region include which amino acid residue clearly mark.Amino acid as specifically described the boundary for constituting a region herein Residue, which is found between each species, to be all conservative and is therefore suitble to be bound region.

It will be understood by those skilled in the art that the invention particularly relates to the SEQ ID NO corresponding to (i) fibrin ferment:1 Gly- Between 427 and Asp-462, between preferably His-450 and Asp-462, between more preferable His-450 and Leu-459, (ii) blood coagulation The SEQ ID NO of factor XI, plasma thromboplastin antecedent a:Between 2 Val-463 and Asp-480, between preferably His-469 and Asp-480 or Ser-477, (iii) the SEQ ID NO of trypsase:Between 3 Leu-73 and Asp-107, preferably His-96 and Asp-107 or Leu-104 Between and (iv) urokinase-type plasminogen activator SEQ ID NO:Between 4 Val-237 and Asp-275, preferably Between Phe-254 or Val-256 and Asp-275 or Asn-274, between more preferable His-262 and Asp-275 or Asn-274 The amino acid in the amino acid residue region in amino acid residue region forms.Therefore, it will be understood by those skilled in the art that egg of the present invention The amino acid sequence of white residue part is alterable, and condition is that the albumen remains, or can be activated as to serine stretch protein Enzyme inhibitor, preferably direct fibrin ferment, plasma thromboplastin antecedent a, trypsase or urokinase-type plasminogen activator inhibitor The serine protease polypeptide that sensibility reduces.Therefore the remainder of the albumen of the present invention can be as it be for example in inhomogeneity Change and change between other serine protease polypeptide.

Corresponding to the amino acid residue quantity in the region in region according to the present invention different plant species serine stretch protein It is conservative between enzyme polypeptide, especially between belonging to mammal group or belonging to each species of primate group.Therefore, ammonia Base acid residue quantity is also conservative in serine protease polypeptide.Corresponding to SEQ ID NO:1 Gly-427 and Asp- Described in the amino acid residue region in the region between 462 guard amino acid residue quantity be 34, include Gly-427 and Asp-462.Corresponding to SEQ ID NO:It is protected described in the amino acid residue region in the region between 1 His-450 and Asp-462 The quantity for the amino acid residue kept is 11, does not include His-450 and Asp-462.For SEQ ID NO:2,3 and 4, it is applicable in identical Principle.

It has been found that being inserted into the amino acid residue region corresponding to the region in albumen of the present invention as described herein At least one amino acid residue obtains the serine protease polypeptide of catalytic activity, but to the suppression of serpin The sensibility of system reduces.

Preferably, it is inserted at least one amino acid residue in amino acid residue region as described herein and replaces at least One amino acid residue is combined.

It includes 1-50, preferably 1-40, more preferable 1-30 and most preferably 1-20 amino to be particularly preferably wherein inserted into The albumen of the present invention of sour residue.In the SEQ ID NO corresponding to fibrin ferment:1 His-450 and Asp-462 or Leu-459 it Between, the SEQ ID NO of plasma thromboplastin antecedent a:Between 2 His-469 and Asp-480 or Ser-477, the SEQ ID of trypsase NO:Between 3 His-96 and Asp-107 or Leu-104 or the SEQ ID NO of urokinase-type plasminogen activator:4 Insertion in the amino acid residue region in the region between His-262 and Asp-275, Asn-274 or Ala-271 preferably include to Few 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19 or 20, more preferable 4 or 5 amino acid Residue, or by least 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19 or 20, more preferable 4 A or 5 amino acid residue compositions.Particularly preferably in the SEQ ID NO corresponding to fibrin ferment:1 His-450 and Asp- It is inserted at least four amino acid residue in the amino acid residue region in the region between 462 or Leu-459, such as is inserted into 8 amino Sour residue.In addition it is particularly preferred that in the SEQ ID NO corresponding to plasma thromboplastin antecedent a:2 His-469 and Asp-480 or Insertion at least five amino acid residue in the amino acid residue region in the region between Ser-477, such as 9 amino acid of insertion are residual Base.In addition it is particularly preferred that in the SEQ ID NO corresponding to trypsase:3 His-96 and Asp-107 or Leu-104 it Between region amino acid residue region in be inserted at least five amino acid residue, such as be inserted into 9 or 11 amino acid residues. In addition it is particularly preferred that in the SEQ ID NO corresponding to urokinase-type plasminogen activator:4 His-262 and Asp- 275, it is inserted at least three amino acid residue in the amino acid residue region in the region between Asn-274 or Ala-271, such as inserts Enter 7 amino acid residues.It will be understood by those skilled in the art that amino acid residue can be corresponding to amino acid as herein defined Any position in the amino acid residue region of full region is inserted into.The amino acid residue being suitably inserted into is selected from as listed in table 1 The group of 20 kinds of naturally occurring amino acid residues.It will be understood by those skilled in the art that the amino acid residue of the insertion can be subjected to In vivo or in vitro chemical modification after translation.As herein above pointed, synthetic DNA, PCR skills can be used in those skilled in the art Art and molecular cloning have to obtain to including 1-50 in the amino acid residue region corresponding to region as herein defined The recombinant dna construct for the DNA sequence dna that the albumen of the present invention of the insertion of a amino acid residue is encoded.

Insertion in the amino acid residue region corresponding to amino acid residue region is preferred：In the SEQ ID of fibrin ferment NO:Between 1 Trp-455 and Arg-456；In the SEQ ID NO of plasma thromboplastin antecedent a:Between 2 Met-474 and Ala-475； In the SEQ ID NO of trypsase:Between 3 Asp-100 and Arg-101, in the SEQ of urokinase-type plasminogen activator ID NO:Between 4 Thr-269 and Leu-270, or corresponding to non-human thrombin, plasma thromboplastin antecedent a, trypsase or urine Between two amino acid residues of these amino acid residues in kinases activator of plasminogen polypeptide.

Particularly preferably include insertion and the amino acid residues of 1-30, preferably 1-8 of at least one amino acid residue Replace the albumen of the present invention being combined.The replacement preferably includes 1,2,3,4,5,8,9,10,11,12,13,14,15,16,17, 18,19,20,21,22,23,24,25,26,27,28,29 or 30, preferably 5-8 amino acid residue, or preferably by 1,2, 3,4,5,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 or 30, It is preferred that 5-8 amino acid residue composition.In the SEQ ID NO corresponding to fibrin ferment:1 His-450 and Asp-462 or Leu- Amino acid residue is replaced in the amino acid residue region in the amino acid residue region between 459 preferably includes 7 or 8 amino acid Residue, or be made of 7 or 8 amino acid residues.In the SEQ ID NO corresponding to plasma thromboplastin antecedent a:2 His-469 with Amino acid residue is replaced in the amino acid residue region in the amino acid residue region between Asp-480 or Ser-477 preferably includes 5 A or 6 amino acid residues, or be made of 5 or 6 amino acid residues.In the SEQ ID NO corresponding to trypsase:3 His-96 and Asp-107 or Leu-104 between amino acid residue region amino acid residue region in replace amino acid it is residual Base preferably includes 7 amino acid residues, or is made of 7 amino acid residues.It is activated corresponding to urokinase-type plasminogen The SEQ ID NO of object:Ammonia is replaced in the amino acid residue region in the amino acid residue region between 4 His-262 and Asp-275 Base acid residue preferably includes 4 or 7 amino acid residues, or is made of 4 or 7 amino acid residues.

Amino acid residue present in region corresponding to the region as herein defined of albumen of the present invention is preferably by table 1 Any one of listed amino acid residue is replaced, preferably by same as shown in " pendant polar " and " side-chain charges " row in table 1 A kind of amino acid substitution.Preferably, SEQ ID NO:In 1 in 451-455 and the 456-458 amino acid residue one A or multiple, SEQ ID NO:One or more of 470-474 and 475-476 amino acid residue, SEQ ID in 2 NO:One or more of 97-100 and 101-103 amino acid residue, SEQ ID NO in 3:262-269 in 4 One or more of a and 270-274 amino acid residue or its corresponding amino acid in non-human serine protease The selected different aminoacids residue from amino acid residue as shown in Table 1 of residue is replaced.

It will be understood by those skilled in the art that when amino acid residue corresponding to the present invention non-human serine protease as When being replaced in the amino acid residue region in region as defined herein, in only preferred albumen of the present invention there is no that A little amino acid residues are preferably replaced.It will be understood by those skilled in the art that only to amino acid residue in specified amino Just SEQ ID NO as mentioned above in the case that replacement in sour full region is illustrated.Therefore he can have Which or more amino acid can be replaced in non-human serine protease which other amino acid residue or which The instruction of other amino acid residues.The present invention relates to all possible combinations of above-mentioned insertion and replacement.

The albumen of the present invention can further comprise at least one amino acid residue corresponding to region as herein defined Amino acid residue region in missing, condition be when and the amino acid residue in the region of natural serine protease polypeptide When quantitative comparison, total amino acid residue quantity is being inserted at least one amino acid residue and is being lacked at least one in the region Increase after amino acid residue.Particularly preferably have at least 1,2,3,4,5,6,7,8,10,15,20 or 30 amino acid residual The albumen of the present invention of the missing of base.

A kind of preferred albumen of the present invention includes the combination of insertion and replacement, or the group be inserted into, replace and/or lacked It closes.Insertion and missing can occur independently of one another, and therefore may be corresponding to amino acid residue region as herein defined There is the insertion of such as 5 amino acid residues and lacking for 4 amino acid in amino acid residue region at different amino acid positions It loses, total amino acid residue quantity in the serine protease polypeptide to increase the present invention.It should be understood to the one skilled in the art that be inserted into or Missing can change the numbering amino acid residues in albumen.

The albumen of the present invention is most preferably included in the SEQ ID NO of fibrin ferment:1 amino acid residue His-450 and Asp- Between 462 or in the SEQ ID NO corresponding to non-human thrombin:Have between the amino residue of 1 His-450 and Asp-462 There are amino acid sequence SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15 or SEQ ID NO:16 amino acid residue region.

The albumen of the present invention is most preferably included in the SEQ ID NO of plasma thromboplastin antecedent a:2 amino acid residue His-469 with Between Asp-480 or in the SEQ ID NO corresponding to non-human factor XIa:The amino of 2 His-469 and Asp-480 There is amino acid sequence SEQ ID NO between residue:7 or SEQ ID NO:8 amino acid residue region.

The albumen of the present invention is most preferably included in the SEQ ID NO of trypsase:3 amino acid residue His-96 and Asp- Between 107 or in the SEQ ID NO corresponding to inhuman trypsase:Between the amino residue of 3 His-96 and Asp-107 With amino acid sequence SEQ ID NO:9 or SEQ ID NO:10 amino acid residue region.

The albumen of the present invention is most preferably included in the SEQ ID NO of urokinase-type plasminogen activator:4 amino acid is residual Between base His-262 and Asp-275 or in the SEQ ID NO corresponding to inhuman urokinase-type plasminogen activator:4 There is amino acid sequence SEQ ID NO between His-262 and the amino residue of Asp-275:11 or SEQ ID NO:12 amino Sour full region.

Present invention also contemplates that substantially with albumen homology of the present invention and the albumen of biology equivalence.The albumen of the present invention is preferred With with SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3 or SEQ ID NO:4 or be more than 90% with its activated form Homology amino acid sequence, wherein the albumen is catalytic activity, or after processing/activation be catalytic activity , and with to serpin, preferably direct thrombin class, blood coagulation FXIa classes, trypsase or urokinase-type The sensibility of the reduction of plasminogen activator inhibitor.

Alternative albumen of the present invention is the recombinant protein for including serine protease polypeptide, and the serine protease is more Peptide is corresponding to SEQ ID NO:Replacement or substitution with amino acid residue on the amino acid residue position of 1 Ile-542, Described in serine protease polypeptide be not Factor X polypeptides or its natural process form or activated form.For example, technology people Member understands, SEQ ID NO:1 position Ile-542 corresponds to SEQ ID NO:2 position His-552, SEQ ID NO:3 position Set Gly-177 and SEQ ID NO:4 position Ser-353.Technical staff is more corresponding to alternative serine protease SEQ ID NO in peptide:1 Ile-542, SEQ ID NO:2 His-552, SEQ ID NO:3 Gly-177 or SEQ ID NO:There is no difficulties in the identification of the amino acid residue position of 4 Ser-353.Preferably, it replaces in following amino acid residue position Set generation：SEQ ID NO:1 Ile-542, SEQ ID NO:2 His-552, SEQ ID NO:3 Gly-177 or SEQ ID NO:4 Ser-353.It replaces or substitution is mutation, preferably conservative or non-conservative mutations.Preferably as replacement Amino acid residue can be any one of amino acid residue as shown in table 1.It is highly preferred that the amino acid residue as replacement For alanine, serine, phenylalanine or glutamic acid.Preferably, serine protease polypeptide is fibrin ferment (original).Such as this portion The replacement of amino acid residue can be combined with any insertion as described herein in albumen described in point.

As used in the present case, term " to the sensibility of the reduction of serpin " or " to the sensibility of the reduction of the inhibition of serpin " refers to the silk for generating 50% maximum suppression (Ki) and needing The concentration of serine protease inhibitor.For the concentration ratio being somebody's turn to do for natural serine protease polypeptide of the polypeptide of the present invention Concentration higher.The natural serine protease polypeptide is preferably derived from blood plasma or recombination generates.Serine protease inhibits The Ki of agent is preferably by identified below：The albumen of the present invention is carried out using 0.001 to 100 μM of serpins Preincubate (precincubation, pre-incubating) then carries out the experiment for wherein analyzing catalytic activity.

With in the amino acid residue region corresponding to amino acid region as herein defined do not have at least one ammonia The Ki of the natural serine protease polypeptide of the insertion of base acid residue is compared, and the Ki of albumen of the present invention is preferably increased above 2x more preferably increases 50x to 100x, and most preferably increases above 100x.

Invention further provides a kind of nucleic acid molecules comprising the nucleotide sequence of coding albumen of the present invention, preferably DNA sequence dna.It will be understood by those skilled in the art that how to generate the DNA sequence dna and such as the amino acid sequence for encoding albumen of the present invention What is manufactured and is detached the nucleic acid molecules with the DNA sequence dna using commonly known recombinant DNA technology.The sequence of nucleic acid molecules Row preferred pin carries out codon optimization to the expression in the host cell of the present invention.Be conducive to carry out in particular host cell The codon of high level expression uses in this way.

The present invention also provides the expression vectors for including nucleic acid molecules of the present invention.

It is preferable to use recombinant DNA technologies well known by persons skilled in the art to be inserted into expression vector for nucleic acid molecules.In this hair In the case of bright, expression vector instructs expression of the albumen of the present invention in host cell.These expression vectors preferably can be used as attached Body (episome, episomes) or the part as chromosomal DNA is added to be replicated in host cell.In addition, expression vector It preferably includes：(i) strong promoter/enhancer, such as CMV or SV40 promoters；(ii) best translation initiation sequence, such as core Sugared body binding site and initiation codon, preferably KOZAK consensus sequences；And (iii) transcription terminator, when albumen is true Include poly- (A) signal when being expressed in nucleus.Suitable expression vector includes plasmid and viral vectors, such as adenovirus, gland companion With virus and retrovirus.It will be understood by those skilled in the art that expression vector ready for use depends on being used for expressing recombination egg White host cell.The expression vector of the present invention is preferably adapted in the prokaryotic cell including bacterial cell or more preferably existing The nucleic acid molecules of the present invention are expressed in the eukaryotic host cell of such as yeast cells and mammalian cell.Particularly preferably feed Newborn animal expression vector pCMV4.

As one substitute, can will the present invention nucleic acid molecules Insertion Into Host Cell genome in.It is described to be inserted into preferably On locus, or in the region expressed in host cell of nucleic acid molecules for ensuring the present invention.

The present invention further provides a kind of host cells comprising nucleic acid molecules or expression vector according to the present invention.This Invention preferably provides expression nucleic acid molecules of the present invention to generate the host cell of albumen of the present invention.The albumen is in host cell Interior generation, or preferably secreted from host cell.

Suitable host cell for the present invention includes prokaryotic cell and eukaryocyte, such as bacterial cell, yeast are thin Born of the same parents, insect cell, zooblast, mammalian cell, mouse (murine) cell, rat (rat) cell, ovine cells, ape Cell and people's cell.The example of suitable eukaryotic host cell include but not limited to 293 cells of HEK, hamster cell system CHO and BHK-21, mouse host cell NIH3T3, NSO and C127, ape host cell COS and Vero and human host cell HeLa, PER.C6, U-937 and Hep G2.Suitable cell can be obtained from the public source of such as ATCC and Life Technologies. Many rotaring dyeing technologies are well known in the art, see, for example, Graham et al., 1973.Virology 52：456；Green Et al., 2012. " Molecular Cloning:A Laboratory Manual ", CSHL Press；Davis et al., " Basic Methods in Molecular Biology ", 1986, Elsevier；And Chu et al., 1981.Gene 13：197.Ability Field technique personnel are preferably introduced one or more exogenous nucleic acid molecules using the technology as described in these references In suitable host cell.

The host cell of the particularly preferred albumen for being used for generating the present invention is 293 cells of HEK.

The present invention further provides a kind of pharmaceutical compositions comprising albumen and pharmaceutical carrier of the invention or figuration Agent.The pharmaceutical composition of the present invention preferably includes diluent, filler, salt, buffer, stabilizer, solubilizer and this field In it is one or more in known other materials.The feature of carrier will depend on administration route, this is for technicians It is known.In order to reduce potential administration of activated serine protease polypeptide thrombotic risk, pharmaceutical composition of the invention Object is preferably included in the albumen of the present invention just activated after subject's application.

Term " subject (object, subject) " refers to mammal group, preferably people.

In the case of the present invention, term " pharmaceutical composition " refers to the albumen and inert carrier or active carrier of the present invention Combination so that composition is suitble in vivo or in vitro therapeutical uses.

As used herein, term " medicinal (pharmaceutically acceptable, pharmaceutically acceptable) " is Refer to compatible with the Physical and chemical characteristics of albumen of the present invention and the validity of the bioactivity of the albumen will not be interfered Non-toxic material.

The enteral administration that may make the pharmaceutical composition suitable composition of the present invention, wherein the composition passes through alimentary canal It absorbs, such as orally ingestible or rectally.The composition is preferably by for example liposomal encapsulated, to prevent proteolysis from dropping Solution.

Preferably so that the present invention pharmaceutical composition be suitble to parenteral administration, wherein the composition pass through it is intravenous, dynamic Arteries and veins is interior, subcutaneous and/or intramuscular introducing.Parenteral administration is related to injecting or be infused into bodily tissue by the pharmaceutical composition of the present invention Or in body fluid, thus it is preferable to use syringe, syringe needle or conduits.Alternatively, needle-less high pressure administration can be used to be used as stomach The method of external administration.

For Injectable composition (such as intravenous composition), carrier can be aqueous or oily solution, dispersant, breast Agent and/or suspending agent.Preferably, carrier is aqueous solution, sterile water, brine, the buffered saline (buffered preferably distilled ) or another pharmaceutical excipient for injection saline.

The pharmaceutical composition of the present invention of fibrin ferment or plasma thromboplastin antecedent a albumen including the present invention can locally apply to for example In wound or wound, or it is administered to blood vessel, preferably the artery of injured area supply blood.The local administration be outside to Medicine, such as with creme (paste, cream), foaming agent, gelling agent, lotion or ointment (ointment agent, ointment, ointment) Form) or parenteral administration, such as by injecting or being transfused, to generate topically or systemically curative effect.For generating part The topical administration of the albumen of the present invention of effect can reduce the risk of potential systemic thrombotic phenomena.

The pharmaceutical composition of the present invention is preferred in various treatment uses.E.g., including fibrin ferment or solidifying of the invention The pharmaceutical composition of blood factor XIa albumen can normal coagulation is damaged wherein disease (such as hemophilia A and B, including blood Friendly disease A and B inhibitor patient group) treatment or improvement in be used as bypass reagent (bypassing agent).Hemophilia A and B suppressions Preparation patient is the patient for having generated the antibody for the product for being used for treating or preventing bleeding episodes.

Therefore the purposes the present invention also provides albumen according to the present invention or pharmaceutical composition as drug.

The present invention further provides according to the present invention including fibrin ferment according to the present invention or plasma thromboplastin antecedent a polypeptides Albumen or pharmaceutical composition according to the present invention including fibrin ferment or plasma thromboplastin antecedent a polypeptides are for complete in subject The method for fully or partially reversing the blood coagulation resisting function of Coagulative inhibitors agent.

Term " blood coagulation resisting function " refers to curative effect, such as the anti-Trostin M due to Coagulative inhibitors agent effect.

The present invention further provides the albumen of the present invention including fibrin ferment or plasma thromboplastin antecedent a polypeptides be used to prepare for The purposes of the drug of the blood coagulation resisting function of Coagulative inhibitors agent is reversed in subject completely or partially.

The present invention further provides the method for the blood coagulation resisting function for reversing Coagulative inhibitors agent completely or partially in subject, The method includes the egg of the present invention including fibrin ferment or plasma thromboplastin antecedent a polypeptides of therapeutically effective amount is applied to the subject The white or pharmaceutical composition of the present invention including fibrin ferment or plasma thromboplastin antecedent a polypeptides.Preferably, method of the invention is used for It prevents or improves and the treatment-related hemorrhage complication of anticoagulant.

As used herein term " therapeutically effective amount " mean the activity that contains in pharmaceutical composition to be administered at The amount divided is enough to realize expected purpose, for example, especially in this case, it is sufficient to reverse the anti-of Coagulative inhibitors agent completely or partially Hemoglutination.Active constituent in pharmaceutical composition according to the present invention, i.e., the amount of albumen of the invention is preferably in about 5mg to 10 In the range of gram albumen.

Therapeutically effective amount may depend on the albumen mean concentration in the blood for the people for needing albumen of the present invention.For example, treatment Effective quantity (i) is preferably 5mg to 10g, preferably 150mg to 10 grams of fibrin ferment according to the present invention；(ii) 5mg to 600mg, excellent Select 5mg to 300mg plasma thromboplastin antecedent a according to the present invention；(iii) 100 micrograms are to 7mg trypsase according to the present invention；With And (iv) 0.004mg to 0.3mg urokinase-type plasminogen activators according to the present invention.It should be understood to the one skilled in the art that according to this The respective dosage of albumen of invention can be different, this is because the normal plasma levels of these albumen or serum levels are different.

Include the medicine according to the present invention of albumen of the present invention (it includes the fibrin ferment or plasma thromboplastin antecedent a polypeptides of the present invention) Compositions preferably to need completely or partially reverse Coagulative inhibitors agent blood coagulation resisting function subject application only 1 time, 2 times or 3 times, preferably only 1 time.

The present invention further provides the albumen of the present invention of the trypsase polypeptide including the present invention or including egg of the present invention The pharmaceutical composition of the present invention of (it includes the trypsase polypeptide of the present invention) for reversing completely or partially in subject in vain The method that the peptide bond hydrolysis of trypsin inhibitor inhibits.

In the same circumstances, the present invention provides the peptide bond water for reversing trypsin inhibitor completely or partially in subject The method inhibited is solved, the method includes the trypsase polypeptide for including the present invention of therapeutically effective amount is applied to the subject Albumen of the present invention or include the albumen of the present invention trypsase polypeptide of the present invention (it include) pharmaceutical composition of the present invention Object.Preferably, the subject is treated before the albumen of the application present invention with trypsin inhibitor.

In the same circumstances, present invention offer includes the albumen of the present invention of trypsase polypeptide according to the present invention for making It is ready for use on the purposes for the drug for reversing the peptide bond hydrolysis of trypsin inhibitor to inhibit completely or partially in subject.

The present invention further provides the albumen of the present invention including trypsase polypeptide to reverse trypsase completely or partially Non-therapeutic use in the peptide bond hydrolysis inhibition of inhibitor.

The present invention further provides the albumen of the present invention of the urokinase-type plasminogen activator including the present invention to be used for Antifibrinolysis is reversed to act on completely or partially in subject, it is preferable that the antifibrinolysis effect is by all As the inhibitor of urokinase-type plasminogen activator inhibitor induces.Preferably, in this case, antifibrinolysis Effect can reduce the destruction of blood clot, preferably in thrombosed such as serious or a large amount of deep vein thrombosis shape At, the intravenous or dialysis intubation of pulmonary embolism, myocardial infarction or blocking.It may further be preferable that urokinase-type plasminogen Activator inhibitor is Mesupron.

The subject is preferably the cancer patient treated with Mesupron.Usually such as to cancer patient's application The urokinase-type plasminogen activator inhibitor of Mesupron is the tissue degradation that can promote transfer in order to prevent.But it applies Antifibrinolysis effect can be generated with the urokinase-type plasminogen activator inhibitor of such as Mesupron, can be used Recombinaton urokinase type plasminogen activator treatment according to the present invention.

Other than other administration routes as described herein, urokinase-type plasminogen activator of the invention can also match It is made and is used for intrapleural administration, to for example improve the discharge of concurrent pleural effusion and empyema.

In the same way, the present invention provides the albumen of the present invention for the urokinase-type plasminogen activator for including the present invention It is used to prepare the drug for reversing antifibrinolysis to act on completely or partially in subject, it is preferable that the anti-fibre Fibrillarin dissolution is induced by the inhibitor of such as urokinase-type plasminogen activator inhibitor.

In the same way, the present invention provides reverse antifibrinolysis to act on completely or partially in subject Method, it is preferable that the antifibrinolysis acts on the inhibitor by such as urokinase-type plasminogen activator inhibitor Induction includes urokinase-type plasminogen activator polypeptide the method includes apply therapeutically effective amount to the subject Albumen of the present invention or the pharmaceutical composition of the present invention for including urokinase-type plasminogen activator polypeptide.Preferably, of the invention Method be used for preventing or improving and the relevant thrombus complication of antifibrinolysis inhibitor therapy.

Alternatively, the present invention provides the recombinant proteins including serine protease polypeptide, and the polypeptide is in outer surface Peptide structure includes the insertion of at least one amino acid residue, wherein the serine protease polypeptide is not that Stuart factor is more Peptide or its catalytic activity form or natural process form, such as factor Xa polypeptide.

Preferably, the peptide structure corresponds to SEQ ID NO:Amino acid between 1 His-450 and Asp-462 is residual The amino acid residue region of base region.In this case, term " correspondence " is used to refer to fibrin ferment and non-fibrin ferment serine egg Amino acid residue region in white enzyme.Alternatively, serine protease polypeptide is selected from by fibrin ferment, plasma thromboplastin antecedent a, pancreas egg The group of white enzyme and urokinase-type plasminogen activator composition, wherein outer surface peptide structure are as noted herein.

In the case of the present invention, if a kind of albumen (possibility) is procoagulant serine protease and if described The full length amino acid sequence of albumen includes corresponding to the amino acid residue segment guarded between the blood coagulation FX factors of different plant species Individually amino acid residue amino acid residue segment or individual amino acid residue, then the albumen be exactly blood coagulation FX or FXa polypeptides.E.g., including it is arrived containing amino acid residue Cys-246 to Ala-250, the Phe-260 corresponding to human blood coagulation X The amino acid residue segment of Leu-266 and/or Asp-413 to His-423 (referring to Genbank accession number AAH46125.1) it is more The procoagulant serine protease of peptide is considered as blood coagulation FXa polypeptides.As discussed herein above, pointed His and Asp ammonia Base acid residue is conservative between different serine protease polypeptides and between different plant species.

For the sake of clear and brief description, each feature is described herein as the part of identical or different embodiment, but It should be understood that the scope of the present invention may include the embodiment of the combination with all or part of feature.

SEQ ID NO:1 (people's blood coagulation factor albumen)

SEQ ID NO:2 (human blood coagulation XI albumen)

SEQ ID NO:3 (- 1 albumen of people's trypsase)

SEQ ID NO:4 (human urokinase type plasminogen activators)

SEQ ID NO：5

1 kkfvppqkay kfdlaaldr

SEQ ID NO：6

1 ppqkaykfdl aaldr

SEQ ID NO：7

1 kkfvppqkay kfdlaasgy

SEQ ID NO：8

1 ppqkaykfdl aasgy

SEQ ID NO：9

1 kkfvppqkay kfdlaalnn

SEQ ID NO：10

1 kkfvppsqef yekfdlvsln n

SEQ ID NO：11

1 kkfvppqkay kfdlaahhn

SEQ ID NO：12

1 ppqkaykfdl aahhn

SEQ ID NO：13

1 pryvppqkay kfdlaaldr

SEQ ID NO：14

1 tkfvppnyyy vhqnfdrval dr

SEQ ID NO：15

1 pkyhqgsgpi lprrtldr

SEQ ID NO：16

1 prydsissky lkellekpld r

Description of the drawings

Block (small figure, panel) shows human thrombin original (SEQ ID NO at the top of Fig. 1：1) in from His-450 to The amino acid residue region of Asp-462 and in people FXI (SEQ ID NO：2), people's trypsase (SEQ ID NO：And human urine 3) Kinases activator of plasminogen (SEQ ID NO：4) corresponding to the arrangement in the amino acid residue region in the region in (alignment).Three blocks show the region of His-450 to Asp-462, people in corresponding to human thrombin original below Region and urine in region, trypsase in FXI between His-469 and Asp-480 between His-96 and Asp-107 are swashed There is the new of insertion in the amino acid residue region in the region in enzyme activator of plasminogen between His-262 and Asp-275 ' A ' and ' B ' albumen change of the human thrombin original, people FXI, people's trypsase and human urokinase type plasminogen activator of generation Body.Conserved residues histidine and asparatate highlight.

Fig. 2 show the crystal structure (PDB 1DWC) of argatroban-antithrombin complex.Argatroban-contact residues (His57、Tyr60A、Lys60F、Leu99、Ile174、Glu192、Ser195、Asp189、Glu192、Gly216、Gly218、 Gly226；Chymotrypsin protein enzyme number (Bode, W. et al. 1989.EMBO J 8：3467-3475)) and Catalytic triad residues His57, Ser195 and Asp102 are shown as rodlike.His91 rings highlight and indicated with arrows.

Fig. 3 show the crystal structure (PDB 4X6P) of compound 33-FXIa compounds.Compound 33 is shown as rodlike Model, FXIa are shown with surface image.Contact residues (His40, Leu41, Cys42, Cys58, Tyr58b, Tyr143, Ile151、Asp189、Lys192、Gly193、Asp194、Ser195、Gly216、Gly218、Tyr228；Chymotrypsin is compiled Number (Bode, W. et al. 1989.EMBO J 8：3467-3475)), Catalytic triad residues His57, Ser195 and His91 rings are prominent Go out display.The latter is in addition also indicated with arrows.

Fig. 4 show the crystal structure (PDB1K1P) of melagatran-trypsase compound.Contact residues (Asp189, Ser190, Gly216 and Gly21；Chymotrypsin protein enzyme number (Bode, W. et al. 1989.EMBO J 8：3467-3475)) and Catalytic triad residues His57, Ser195 are shown as rodlike.His91 rings highlight and indicated with arrows.

Fig. 5 are in human thrombin original (' fibrin ferment ', SEQ ID NO:1) and in corresponding to human thrombin original His-450 is arrived In the amino acid residue region in the region of Asp-462 have be inserted into newly generated human thrombin original protein variant (' ISO1 ', ' ISO2 ', ' NSC ', ' KL10 ', ' ALB ') in amino acid residue region from His-450 to Asp-462 arrangement.Positioned at position Conserved residues histidine at 450 and the asparatate at position 462 highlight.

Inhibition-series 1 of Fig. 6 direct thrombin inhibitors dabigatrans to the thrombin activity that develops the color.Increasing concentration In the presence of the dabigatran of (20nM-20 μM), 5nM derives from the fibrin ferment (' IIa ' of blood plasma；Block A), activation derive from The factor (' pd-IIa ' of blood plasma；Block B), recombination fibrin ferment (' r-IIa '；Block C), recombination blood coagulation enzyme variants ISO1 (‘ISO1’；Block D) or recombination blood coagulation enzyme variants NSC (' NSC '；Block E) peptidyl (peptidyl) substrate conversion efficiency (S- 2238；100μM).IC50 concentration converts S-2238 by nonlinear regression by using Graphpad Prism software suites Rate (mOD/min) is fitted acquisition.All data points represent the average value of two independent experiments.Block F：Substrate conversion efficiency (speed) is plotted as the incubation rate there is no inhibitor.It is clearly illustrated in block F, each variant shows ratio Compare higher normalizated velocity.

Inhibition-series 2 of Fig. 7 direct thrombin inhibitors dabigatrans to the thrombin activity that develops the color.Increasing concentration In the presence of the dabigatran of (20nM-20 μM), 5nM derives from the fibrin ferment (' IIa ' of blood plasma；Block A), recombination fibrin ferment (‘r-IIa’；Block B), recombination fibrin ferment modification A LB (' ALB '；Block C), recombination blood coagulation enzyme variants KL10 (' KL10 '；Block D) or recombination blood coagulation enzyme variants ISO2 (' ISO2 '；Block E) peptide based substrate conversion ratio (S-2238；100μM).IC50 concentration S-2238 conversion ratios (mOD/min) are fitted by nonlinear regression by using Graphpad Prism software suites and are obtained .All data points represent the average value of two independent experiments.Block F：Substrate conversion efficiency (speed) be plotted as there is no Incubation rate in the case of inhibitor.It is clearly illustrated in block F, each variant is shown than compareing higher normalization speed Degree.

Crystal structure (PDB 1KTS) (Hauel et al., J Med of fibrin ferment in the compound of Fig. 8 and dabigatran Chem 45：1757-1766(2002)).Shown in bar graph active-site residues His406, Asp462 and Ser568 with And dabigatran interaction residue Tyr410, Leu459, Ile542, Asp562, Trp590 and Gly591.The S4 of active site The position of secondary bag (subpocket) is marked by ellipse, and indicates residue Ile542.

Inhibition-series 3 of Fig. 9 direct thrombin inhibitors dabigatrans to the thrombin activity that develops the color.Increasing concentration In the presence of the dabigatran of (20nM-20 μM), 5nM from blood plasma fibrin ferment (block A), recombination fibrin ferment (block B), Recombinate blood coagulation enzyme variants I542F (block C), recombination blood coagulation enzyme variants I542A (block D), (areas recombination blood coagulation enzyme variants I542E Block E) or recombination blood coagulation enzyme variants I542S (block F) peptide based substrate conversion ratio (S-2238；100μM).IC50 concentration passes through Acquisition is fitted to S-2238 conversion ratios (mOD/min) by nonlinear regression using Graphpad Prism software suites. All data points represent the average value of two independent experiments.

Inhibition of the normalized direct thrombin inhibitor dabigatrans of Figure 10 to the thrombin activity that develops the color.It is dense increasing It spends in the presence of the dabigatran of (20nM-20 μM), fibrin ferment (' IIa '), recombination fibrin ferment (' r-s of the 5nM from blood plasma IIa '), recombination blood coagulation enzyme variants I542A, recombination blood coagulation enzyme variants I542E, recombination blood coagulation enzyme variants I542F or recombination blood coagulation Peptide based substrate conversion ratio (the S-2238 of enzyme variants I542S；100μM).Substrate conversion efficiency (speed) is plotted as there is no inhibition Incubation rate in the case of agent.All data points represent the average value of two independent experiments.It clearly illustrates, each variant is equal It shows than compareing higher normalizated velocity.

Figure 11 human thrombin originals (' fibrin ferment ', SEQ ID NO:1) in from Cys-536 to Cys-550, people's factor XI, plasma thromboplastin antecedent (' FXIa ', SEQ ID NO:2) in from Cys-545 to Cys-560, people's trypsase -1 (' trypsase ', SEQ ID NO:3) In from Cys-171 to Cys-185 and human urokinase type plasminogen activator (' uPA ', SEQ ID NO:4) from Cys- in The arrangement in 345 to Cys-361 amino acid residue region.Residue Ile-542 (SEQ ID NO:1)、His-552(SEQ ID NO:2)、Gly-177(SEQ ID NO:And Ser-353 (SEQ ID NO 3):4) it highlights.

Table 1

Embodiment

Embodiment 1：Material and method

Material：Direct serpin is obtained from Alsachim and Adooq, and corn trypsin inhibitor comes From Haematologic Technologies.FXI- is lacked and the human plasma Neoplastin CI plus of factor-missing The APTT of 10 and TriniCLOT automations is obtained from Diagnostica Stago.Peptide based substrate S-2238, S-2366, S- 2444 and S-2222 is obtained from Chromogenix.All Tissue culture reagents are all from Life Technologies, in addition to pancreas Island element-transferrins-sodium selenite (ITS) comes from Roche.Calibrator and fluorogenic substrate (FluCa) come from Thrombinoscope BV.By the egg L- phosphatidyl cholines of 75% (w/w) and the pig brain L- phosphatidyl silks of 25% (w/w) The small single layer phospholipid capsule bubble (PCPS) of propylhomoserin (Avanti Polar Lipids) composition is prepared and is characterized as previously described (Higgins et al. 1983.J Biol Chem 258：6503-6508).The general production technology and purification technique of recombinant protein As described in " the Molecular Cloning " in Green and Sambrook July the 4th edition in 2012.

The expression and purifying of fibrin ferment：Using LipofectAMINE 2000 (Invitrogen) and pSV2neo as can Coding factor modification A (is had SEQ ID NO by selected marker plasmid between His-450 and Asp-462:5 amino Factor (the SEQ ID NO of acid sequence:1)) and B (has SEQ ID NO between His-450 and Asp-462:6 amino Factor (the SEQ ID NO of acid sequence:1)) and the plasmid of wild type factor (pcDNA3.1 (+)) introduces HEK In 293 cells.High-expression clone is substantially such as (Orcutt et al. 2004.J Biol Chem 279：It is 54927-54936) described ELISA and PT setting tests based on factor specificity are selected.It is 10- layer cell factories by selected clonal expansion (Nalge-Nunc, Naperville, IL), and improved with the Dulbecco of 5 μ g/ml ITS and 10 μ g/ml vitamin Ks supplement Eagle's medium/F-12 culture mediums in cultivate.Adjusted culture medium is collected 5-6 days, centrifugation, and in 1mM benzenecarboximidamides In the presence of stored at -20 DEG C.Factor is substantially such as (Orcutt et al. 2004.J Biol Chem 279：54927- 54936) described using Q- Ago-Gels FF (GE Healthcare), HQ POROS matrix (Affinity Biologicals) and ceramic hydroxyapatite matrix (Bio-Rad) is purified from adjusted culture medium.The fibrin ferment of purifying Original is stored at -20 DEG C in the HBS containing 50%vol/vol glycerine.Purity of protein uses prefabricated 4- under the reducing conditions 12% gradient gel (Invitrogen) is assessed by SDS-PAGE, is then dyed with coomassie brilliant blue R_250.Such as (Lundblad et al., 1976.Methods Enzymol 45：156-176) it is described prepared activation carried out to factor after Fibrin ferment is purified.

The expression and purifying of FXI：Using LipofectAMINE 2000 (Invitrogen) and pSV2neo as may be selected Coding FXI modification As (are had SEQ ID NO by marker plasmid between His-469 and Asp-480:7 amino acid sequence FXI(SEQ ID NO:2)) and B (has SEQ ID NO between His-469 and Asp-480:The FXI of 8 amino acid sequence (SEQ ID NO:2)) and the plasmid of wild type FXI (pcDNA3.1 (+)) is introduced into newborn hamster kidney (BHK) cell, the matter Grain (pcDNA3.1 (+)) is merged with HPC4- antibody recognition sequences (amino acid sequence EDQVDPRLIDGK) to promote by its end C- Into purifying.High-expression clone is substantially such as (Toso et al. 2004.J Biol Chem 279：21643-21650) it is directed to factor Ⅴ It is described to be selected based on FXI- specific ELISAs and APTT setting tests.Selected clonal expansion is to three-necked flask (triple Flasks) in (Nalge-Nunc, Naperville, IL), and with 5 μ g/ml ITS and 1.0mg/ml Albumax (Invitrogen) it is cultivated in Eagle's medium/F-12 culture mediums of the Dulbecco improvement supplemented.Adjusted culture medium It collects 5-6 days, centrifugation, and is stored at -20 DEG C in the presence of 1mM benzenecarboximidamides.Adjusted culture medium thaws at 37 DEG C, Merge, and is loaded into the anti-HPC4 Ago-Gels that 25mM Tris, 0.05M NaCl, 5mM CaCl2 through pH 7.4 are balanced On column.Then the column equilibration buffer solution uses the 25mM Tris, 0.5M NaCl, 5mM EDTA elutions of pH 7.4, then With the 25mM Tris of pH 7.4,2M NaCl, 5mM EDTA elutions.There to be the active fractions of FXI to merge, to (it is directed to, it is right It is anti-, versus) it is dialysed by 20mM Hepes, the 150mM NaCl of the pH 7.4 of ultrafiltration (Millipore) concentration, and will purifying Albumen stores at -80 DEG C.Purity of protein uses prefabricated 4-12% gradient gels (Invitrogen) logical under the reducing conditions It crosses SDS-PAGE to be assessed, then be dyed using coomassie brilliant blue R_250.In such as (Ogawa et al., 2005.J Biol Chem 280：It is 23523-23530) described that FXI purified to factor XI, plasma thromboplastin antecedent a after prepared activation.

The expression and purifying of urokinase-type plasminogen activator (uPA)：Use LipofectAMINE 2000 (Invitrogen) and pSV2neo is as selectable marker plasmid, by encoding human uPA modification As (His-262 and Asp-275 it Between have SEQ ID NO:UPA (the SEQ ID NO of 11 amino acid sequence:4)) (have between His-262 and Asp-275 with B There are SEQ ID NO:UPA (the SEQ ID NO of 12 amino acid sequence:) and the plasmid of wild type uPA (pcDNA3.1 (+)) 4) It is introduced into mammalian cell (HEK 293 is BHK), the plasmid (pcDNA3.1 (+)) is known by its end C- and HPC4- antibody Other sequence (amino acid sequence EDQVDPRLIDGK) or with His- labels (6His：HHHHHH or 12His：HHHHHHHHHHHH) melt It closes to promote to purify.High-expression clone is selected based on people uPA- specific ELISAs (R＆D Systems).By selected clone Expand in three-necked flask (triple flasks) (Nalge-Nunc, Naperville, IL), and with 5 μ g/ml ITS and It is trained in Eagle's medium/F-12 culture mediums of the Dulbecco improvement of 1.0mg/ml Albumax (Invitrogen) supplements It supports.Adjusted culture medium is collected 5-6 days, centrifugation, and is stored at -20 DEG C in the presence of 1mM benzenecarboximidamides.

Substantially thawed at 37 DEG C for culture medium adjusted as described in FXI variants, merge, and using anti- HPC4 agarose gel purifications.Alternatively, the uPA variants of His labels are purified using immobilization metal affinity chromatography.It is purified Albumen stored at -80 DEG C, purity of protein under the reducing conditions use prefabricated 4-12% gradient gels (Invitrogen) It is assessed by SDS-PAGE, is then dyed using coomassie brilliant blue R_250.UPA variants are activated by human plasmin, and are adopted With benzamidine-sepharose gel-purified.

The expression and purifying of trypsase：Using LipofectAMINE 2000 (Invitrogen) and pSV2neo as Coding -1 modification A of human trypsinogen (is had SEQ ID NO by selectable marker plasmid between His-96 and Asp-107:9 Amino acid sequence trypsase (SEQ ID NO:3)) and B (has SEQ ID NO between His-96 and Asp-107:10 Amino acid sequence trypsase (SEQ ID NO:) and wild type trypsase (SEQ ID NO 3):3) plasmid (pcDNA3.1 (+)) is introduced into 293 cells of HEK, and it is scarce to be modified to it for targeting sequencing in the plasmid (pcDNA3.1 (+)) Weary Trypsin cleavage sites (referring to patent EP 1141263A1), and pass through its end C- and HPC4- antibody recognition sequences (amino acid sequence EDQVDPRLIDGK) or with His- labels (6His：HHHHHH or 12His：HHHHHHHHHHHH) fusion is to promote Into purifying.High-expression clone is selected based on human trypsinogen-specific ELISA (MyBioSource).Selected clone expands Open up in three-necked flask (triple flasks) (Nalge-Nunc, Naperville, IL), and with 5 μ g/ml ITS and It is trained in Eagle's medium/F-12 culture mediums of the Dulbecco improvement of 1.0mg/ml Albumax (Invitrogen) supplements It supports.Adjusted culture medium is collected 5-6 days, centrifugation, and is stored at -20 DEG C in the presence of 1mM benzenecarboximidamides.

Substantially thawed at 37 DEG C for culture medium adjusted as described in FXI variants, merge, and using anti- HPC4 agarose gel purifications.Alternatively, the trypsinogen variant of His labels is pure using immobilization metal affinity chromatography Change.Purifying protein stores at -80 DEG C, and purity of protein uses prefabricated 4-12% gradient gels under the reducing conditions (Invitrogen) it is assessed by SDS-PAGE, is then dyed using coomassie brilliant blue R_250.- 1 variant of trypsase is logical It crosses and enterokinase-dependence cutting is carried out to prepare to -1 variant of trypsinogen, and use SP- or benzamidine-sepharose gel Purifying.

Embodiment 2：Direct inhibition of the serpin to serine protease.Originally, peptide based substrate hydrolyzes Dynamics (S-2388, S-2366, S-2444 or S-2222 respectively as fibrin ferment, FXIa, urokinase-type plasminogen activate The specific substrate of object or trypsase) become in above-mentioned fibrin ferment, FXIa, urokinase-type plasminogen activator or trypsase (10-500 μM) measurement of substrate for increasing concentration is used in the presence of body and wild type.Direct serpin combines And the ability of serine protease is inhibited to be tested using the inhibition constant (Ki) of typical Reverse transcriptase by assessing, institute's commentary Estimate the direct inhibitor for increasing concentration by being used under fixed peptide based substrate (in Km or in Km or more) and the concentration of enzyme (1nM-10 μM) measures the hydrolysis initial velocity of peptide based substrate to carry out.All kinetic measurements are pH's 7.5 It is carried out in 20mM Hepes, 0.15M NaCl, 0.1% (w/v) PEG 8000,2mM CaCl2.

Embodiment 3：The generation of fibrin ferment is tested.The generation of fibrin ferment from described in the past scheme (Hemker et al., 2003.Pathophysiol Haemost Thromb 33：4-15) change.Briefly, the generation curve negotiating of fibrin ferment is used Corn trypsin inhibitor (70 μ g/ml), PCPS (20 μM) and substrate buffer solution (Fluca) supplement lack factor or The blood plasma for lacking FXI obtains.The formation of fibrin ferment is become by the fibrin ferment A variants or B for adding 1 unit (specific coagulation activity) (it is mixed with direct serpin in advance for body, FXIa A variants or B variants and wild type fibrin ferment or FXIa Close) start.In alternative setting, the zymogen forms of protein variant are assessed.In doing so, factor-lacks The blood plasma of lose or FXI- missings uses tissue factor (TF (Innovin) is finally 2 or 20pM), the suppression of corn trypsase respectively Preparation (70 μ g/ml), PCPS (20 μM), direct inhibitor and 1 unit (specific coagulation activity) recombination prothrombin activated or FXI variants supplement.The formation of fibrin ferment is started by the way that Fluca to be added in blood plasma.Use Thrombinoscope softwares (Thrombinoscope BV) is measured the formation of fibrin ferment in every 20 seconds in 30 minutes, and is corrected for calibrator. Delay time, average intrinsic coagulation enzyme potential (area under thrombin generation curves), time and the blood coagulation for reaching peak value The generation peak value of enzyme is from least 3 individual experiment calculations.

Embodiment 4：The clot dissolution time of urokinase-type plasminogen activator variant is assessed.Clot dissolution time is basic On assessed (Mosnier et al., 2001.Thromb Haemost 86 as previously described：1035-1039).Briefly, group The factor (TF, Innovin) and PCPS are knitted in the 25mM Hepes of pH 7.4,137mM NaCl, 3.5mM KCl, 0.1%BSA It is incubated 1 hour at 37 DEG C.TF/PCPS mixtures (final is 0.5pM/20 μM) (are finally with blood plasma (50%v/v), tPA 150U/ml) and CTI (being finally 70 μ g/ml) is incubated 10 minutes at 37 DEG C.The Ca2+ of blood coagulation preincubate at 37 DEG C is (most It is 17mM eventually) start.By the way that the absorbance at 405nm is measured at 37 DEG C in SpectraMax M2e microplate reader to grumeleuse It is formed and subsequent dissolving monitors 4 hours.Clot dissolution time is defined as the limpid average value for being changed into muddiness and changes to muddiness For limpid average value, S fittings are carried out to turbidity curve by using Graphpad Prism 5 to determine.

Embodiment 5：In SEQ ID NO:There is the recombination prothrombin activated of insertion in 1 region His450-Asp462.

Material and method

Direct thrombin inhibitor dabigatran is obtained from Alsachim (France).Former from the human thrombin of blood plasma, From the human thrombin (α-fibrin ferment, IIa) of blood plasma, people's factor Xa from blood plasma, people's factor Ⅴ a from blood plasma, And thrombin inhibitor dansyl arginine (dansylarginine) N- (3- ethyl -1,5- pentanes diyl) amide (DAPA) Haematologic Technologies are come from.The human plasma and prothrombin time coagulation of factor-missing try Reagent STA-Neoplastine CI plus 10 are tested to obtain from Diagnostica Stago.Peptide based substrate S-2238 from(Instrumentation Laboratory) is obtained.All Tissue culture reagents are all from Life Technologies (Thermo Fisher Scientific), in addition to Insulin-Transferrin-sodium selenite (ITS) comes from Roche.General production technology and purification technique such as Green and Sambrook July the 4th edition in 2012 of recombinant protein " Molecular Cloning " is described.By the egg L- phosphatidyl cholines of 75% (w/w) and the pig brain L- phosphatide of 25% (w/w) The small single layer phospholipid capsule bubble (PCPS) of acyl group serine (Avanti Polar Lipids, Inc., the U.S.) composition is as described above Prepared and characterized (Higgins et al., J Biol Chem 258：6503-6508(1983)).

Use LipofectAMINE(Invitrogen) by encoding wild type factor (SEQ ID NO:1) And there is (i) SEQ ID NO between His-450 and Asp-462:5 (factor ISO1, from the quasi- cobra in east Homologous region in isotype factor X), (ii) SEQ ID NO:13 (factor ISO2), (iii) SEQ ID NO:14 (blood coagulations Proenzyme NSC, the homologous region in the blacksnake venom factor X of Australia), (iv) SEQ ID NO:15 (factor KL10, sources Homologous region in Human kallikrein 10) or (v) SEQ ID NO:16 (fraction A prothrombin LB derives from human albumin) The plasmid (pcDNA3.1 (+)) of the factor variant of amino acid sequence is introduced into 293 cells of HEK.High-expression clone is substantially Such as Orcutt et al., J Biol Chem, 279：54927-54936 (2004) ELISA based on factor specificity with Factor-time solidification experiment is selected.Selected clonal expansion is to 175cm²Flask in, and with 5 μ g/ml ITS and It is cultivated in Eagle's medium/F-12 culture mediums of the Dulbecco improvement of 10 μ g/ml vitamin Ks supplement.Adjusted culture Base is collected 24 hours, is concentrated in the brine of HEPES- bufferings of pH 7.5 using the 30kDa filters rotated, and at -20 DEG C Under be stored in the glycerine of 50%vol/vol.Factor antigen concentration uses the pairs of antibody for being used for detecting factor ELISA (CL20111K, Cedarlane Laboratories) is measured.Prothrombin activity uses the fibrin ferment of known concentration Original work are that standard uses the blood for lacking factor using the stage prothrombin time coagulation experiment of factor specificity 10 reagents of STA-Neoplastin CI plus in slurry measure.Specific activity (U/mg) from prothrombin activity (U/ml) with The ratio of factor antigen concentration (mg/ml) is derived.

Inhibition of the direct thrombin inhibitor dabigatran to fibrin ferment

By in 5 minutes at ambient temperature in the presence of 10 μM of DAPA using prothrombinase (1nM factors Xa, 50nM factor Ⅴs a, 50 μM of PCPS, 5mM calcium) it is incubated the factor from blood plasma described in the above paragraph (Haematologic Technologies) or recombination wild type factor or recombination prothrombin activated variant (ISO1, ISO2, NSC, KL10 and ALB) (125nM) activation be fibrin ferment.Then sample is quenched in EDTA (being finally 25mM), and containing The EDTA (50mM) of pH 7.5, NaCl (150mM), 0.1%PEG8000 and HEPES (20mM) buffer solution in be diluted to 5nM (final).Direct thrombin inhibitor dabigatran combine and inhibit activation factor (pd-IIa) from blood plasma or The ability of the factor (r-IIa) of the activation of recombination or the factor variant of the activation of recombination is dense by double of maximum suppression Degree (IC50) is assessed to test, the assessment by under fixed S-2238 concentration (100 μM) using increasing concentration (20nM-20 μM) of dabigatran measures the hydrolysis initial velocity of peptide based substrate S-2238 to carry out.It is aobvious to the residual of peptide based substrate Color activity measures in 10 minutes in the microplate reader (SpectraMax M2e, Molecular Devices) for being located at A405nm. IC50 concentration is by using 6 software suites of Graphpad Prism by nonlinear regression to S-2238 conversion ratios (mOD/min) It is fitted acquisition.Using the fibrin ferment (IIa, Haematologic Technologies) from blood plasma as a contrast into The same experiment of row.

As a result

The result of these experiments is shown in table 2 and Fig. 6 and Fig. 7.It can be seen that and claimed from all these results Factor amino acid residue region in insertion provide to the direct thrombin inhibitor of such as dabigatran go it is quick Effect, while still there is solidification possibility or blood coagulation enhancing effect.

Table 2

Table 2 shows the feature of different factor variants.It is expressed as IIa from the fibrin ferment of blood plasma, recombination is wild Type fibrin ferment is expressed as r-IIa.It is solidifying from using a stage factor-time solidification experiment of factor specificity to measure Blood zymogen activity (U/ml) and the ratio of factor antigen concentration (mg/ml) derive specific activity (U/mg).Chomogenic activity Percentage (%) shows the S- with each relevant factor variant of standard curve of the fibrin ferment from blood plasma of purifying 2238 conversion ratios.Half maximum suppression concentration (IC50) display inhibits reaching for the Chomogenic activity needs of 50% 5nM blood coagulation enzyme variants Than the concentration for adding group.

Embodiment 6. is in SEQ ID NO:The recombination replaced or replaced with amino acid residue at 1 position Ile-542 is solidifying Hemase is former.

Material and method

Direct thrombin inhibitor dabigatran is obtained from Alsachim (France).Former from the human thrombin of blood plasma, From the human thrombin (α-fibrin ferment, IIa) of blood plasma, people's factor Xa from blood plasma, people's factor Ⅴ a from blood plasma, And thrombin inhibitor dansyl arginine N- (3- ethyl -1,5- pentanes diyl) amide (DAPA) comes from Haematologic Technologies.The human plasma and prothrombin time coagulation test reagent STA- of factor-missing Neoplastine CI plus 10 are obtained from Diagnostica Stago.Peptide based substrate S-2238 from (Instrumentation Laboratory) is obtained.All Tissue culture reagents are all from Life Technologies (Thermo Fisher Scientific), in addition to Insulin-Transferrin-sodium selenite (ITS) from Roche.Recombination The 4th edition " Molecular of general production technology and purification technique such as Green and Sambrook July in 2012 of albumen Described in Cloning ".By the egg L- phosphatidyl cholines of 75% (w/w) and the pig brain L- phosphatidy serines of 25% (w/w) (Avanti Polar Lipids, Inc., the U.S.) composition small single layer phospholipid capsule bubble (PCPS) as described above carry out prepare and Characterize (Higgins et al., J Biol Chem 258：6503-6508(1983)).

Use LipofectAMINE(Invitrogen) by encoding wild type factor (SEQ ID NO:1) (wherein it is located at SEQ ID NO with factor variant:Different bright amino acid at 1 amino acid residue position 542 is by alanine (I542A, unprotected side chain), serine (I542S, small side chain), phenylalanine (I542F, huge large volume side chain) or glutamic acid (I542E, electrically charged side chain) replace) plasmid (pcDNA3.1 (+)) be introduced into 293 cells of HEK.High-expression clone is basic It is upper such as Orcutt et al., J Biol Chem, 279：ELISA based on factor specificity described in 54927-54936 (2004) It is selected with factor-time solidification experiment.Selected clonal expansion is to 175cm²Flask in, and with 5 μ g/ml ITS It is cultivated in Eagle's medium/F-12 culture mediums of the Dulbecco improvement of 10 μ g/ml vitamin Ks supplement.Adjusted training It supports base to collect 24 hours, be concentrated in the brine of HEPES- bufferings of pH 7.5 using the 30kDa filters rotated, and -20 It is stored in the glycerine of 50%vol/vol at DEG C.Factor antigen concentration uses the pairs of antibody for being used for detecting factor ELISA (CL20111K, Cedarlane Laboratories) is measured.Prothrombin activity uses the factor of known concentration It uses a stage prothrombin time coagulation of factor specificity to test as standard and uses the blood plasma for lacking factor In 10 reagents of STA-Neoplastin CI plus measure.From prothrombin activity (U/ml) and factor antigen concentration (mg/ml) ratio derives specific activity (U/mg).

Inhibition of the direct thrombin inhibitor dabigatran to fibrin ferment

By in 5 minutes at ambient temperature in the presence of 10 μM of DAPA using prothrombinase (1nM factors Xa, 50nM factor Ⅴs a, 50 μM of PCPS, 5mM calcium) be incubated will recombination wild type factor or recombination prothrombin activated variant (I542S, I542A, I542F and I542E) (125nM) activation be fibrin ferment.Then sample is quenched in EDTA (being finally 25mM), and It is diluted in the buffer solution of the EDTA (50mM) containing pH 7.5, NaCl (150mM), 0.1%PEG8000 and HEPES (20mM) To final for 5nM.Direct thrombin inhibitor dabigatran combines and inhibits the factor (r-IIa) or again of the activation of recombination The ability of the factor variant of the activation of group is assessed by double of maximum suppression concentration (IC50) to test, the assessment Pass through (100 μM) (20nM-20 μM) measurement peptide substrates of direct inhibitor using increase concentration under fixed S-2238 concentration The hydrolysis initial velocity of object S-2238 carries out.A405nm's is being located in 10 minutes to the residual Chomogenic activity of peptide based substrate It is measured in microplate reader (SpectraMax M2e, Molecular Devices).IC50 concentration is by using Graphpad Prism 6 software suites are fitted S-2238 conversion ratios (mOD/min) by nonlinear regression to obtain.Using from blood plasma Fibrin ferment (IIa, Haematologic Technologies) carries out identical experiment as a contrast.

As a result

The result of these experiments is shown in table 3 and Fig. 9 and Figure 10.It can be seen that from all these results in SEQ ID NO:1 amino acid residue position Ile-542, which is replaced, replaces or is mutated, can be provided to the direct solidifying of such as dabigatran The desensitization of thrombin inhibitor, while still there is solidification possibility or blood coagulation enhancing effect.

Table 3.

Table 3 shows the feature of factor variant.It is expressed as IIa from the fibrin ferment of blood plasma, recombination wild type is solidifying Hemase is expressed as r-IIa.Specific activity (U/mg) by prothrombin activity (U/ml) and factor antigen concentration (mg/ml) ratio Example derives that the prothrombin activity is determined using a stage factor-time solidification experiment of factor specificity. The percentage (%) of Chomogenic activity shows each relevant fibrin ferment of standard curve with the fibrin ferment from blood plasma of purifying The S-2238 conversion ratios of former variant.Half maximum suppression concentration (IC50) display inhibits the colour developing of 50% 5nM blood coagulation enzyme variants to live Property need dabigatran concentration.

Claims (21)

1. a kind of recombinant protein, including serine protease, the serine protease includes at least in outer polypeptide surface structure The insertion of one amino acid residue；The wherein described serine protease polypeptide is not Factor X polypeptides or its natural process Or activated form.

2. albumen according to claim 1, wherein the peptide structure corresponds to SEQ ID NO:1 His-450 with The amino acid residue region in the amino acid residue region between Asp-462.

3. albumen according to claim 1, wherein the serine protease is selected from fibrin ferment, plasma thromboplastin antecedent a, pancreas egg White enzyme and urokinase-type plasminogen activator；And the wherein described peptide structure is：

The SEQ ID NO of fibrin ferment:Between 1 Gly-427 and Asp-462, between preferably His-450 and Asp-462, more preferably Amino acid residue region between His-450 and Leu-459；

The SEQ ID NO of plasma thromboplastin antecedent a:Between 2 Val-463 and Asp-480, preferably His-469 and Asp-480 or Ser- Amino acid residue region between 477；

The SEQ ID NO of trypsase:Between 3 Leu-73 and Asp-107, preferably His-96 and Asp-107 or Leu-104 it Between amino acid residue region；

The SEQ ID NO of urokinase-type plasminogen activator:Between 4 Val-237 and Asp-275, preferably His-262 with Amino acid residue region between Asp-275 or Asn-274.

4. albumen according to any one of claim 1-3, wherein the insertion includes 1-50, preferably 1-20 amino Sour residue.

5. according to the albumen described in any one of claim 1-4, wherein described be inserted into includes 4 to 50, preferably 4 to 20 ammonia Base acid residue.

6. according to the albumen described in any one of claim 2-5, wherein at least one amino acid is residual in this region The insertion of base is combined with the replacement of at least five amino acid residue.

7. according to the albumen described in any one of claim 2-6, wherein the region have after being inserted into and/or replacing with Lower amino acid sequence：

SEQ ID NO:SEQ ID NO between 1 His-450 and Asp-462:5、SEQ ID NO:6、SEQ ID NO:13、 SEQ ID NO:14、SEQ ID NO:15 or SEQ ID NO:16；

SEQ ID NO:SEQ ID NO between 2 His-469 and Asp-480:7 or SEQ ID NO:8；

SEQ ID NO:SEQ ID NO between 3 His-96 and Asp-107:9 or SEQ ID NO:10；

And/or SEQ ID NO:SEQ ID NO between 4 His-262 and Asp-275:11 or SEQ ID NO:12.

8. a kind of recombinant protein, including serine protease polypeptide, the serine protease polypeptide is corresponding to SEQ ID NO:On the amino acid residue position of 1 Ile-542 there is amino acid residue to replace, wherein the serine protease polypeptide is not It is Factor X polypeptides or its natural process or activated form.

9. a kind of nucleic acid molecules, including encode the DNA sequence dna of the albumen according to any one of claim 1-8.

10. a kind of expression vector, including nucleic acid molecules according to claim 9.

11. a kind of host cell, including nucleic acid molecules according to claim 9 or expression according to claim 10 Carrier.

12. a kind of pharmaceutical composition includes albumen and pharmaceutical carrier according to any one of claim 1-8.

13. the albumen according to any one of claim 1-8 or pharmaceutical composition according to claim 12, are used as Drug.

14. the albumen according to any one of claim 1-8 or pharmaceutical composition according to claim 12, are used for It reverses in subject and is used in the method for the blood coagulation resisting function of Coagulative inhibitors agent, wherein the albumen includes fibrin ferment or blood coagulation Factor XI, plasma thromboplastin antecedent a.

15. a kind of method for the blood coagulation resisting function for reversing Coagulative inhibitors agent in subject, the method includes to described tested Person applies the following terms of therapeutically effective amount：

According to the albumen described in any one of claim 1-8, wherein the albumen includes fibrin ferment or plasma thromboplastin antecedent a, or Person

Pharmaceutical composition according to claim 12, wherein described pharmaceutical composition include containing fibrin ferment or coagulation factor The albumen of XIa.

16. the albumen according to any one of claim 1-8 is used to prepare for reversing Coagulative inhibitors agent in subject Blood coagulation resisting function drug purposes, wherein the albumen includes fibrin ferment or plasma thromboplastin antecedent a.

17. the albumen according to any one of claim 1-8 or pharmaceutical composition according to claim 12, are used for It reverses in subject and is used in the method for the peptide bond hydrolysis inhibition of trypsin inhibitor, wherein the albumen includes tryptose Enzyme, wherein described pharmaceutical composition include trypsase.

18. a kind of method for reversing the peptide bond hydrolysis of trypsin inhibitor to inhibit in subject, the method includes to institute Subject is stated using albumen of the therapeutically effective amount according to any one of claim 1-8 or according to described in claim 12 Pharmaceutical composition, wherein the albumen includes trypsase, wherein described pharmaceutical composition includes the egg containing trypsase In vain.

19. the albumen according to any one of claim 1-8 is used to prepare for reversing trypsase suppression in subject The purposes for the drug that the peptide bond hydrolysis of preparation inhibits, wherein the albumen includes trypsase.

20. the albumen according to any one of claim 1-8 is in the peptide bond hydrolysis for reversing trypsin inhibitor inhibits Non-therapeutic use, wherein the albumen includes trypsase.

21. the albumen according to any one of claim 1-8 or pharmaceutical composition according to claim 12, are used for It is used in reversing antifibrinolysis to act on completely or partially in subject, wherein the albumen includes urokinase-type fibrinolytic Activation of zymogen object.